thapsigargin and Hypertension

Pronounced differences in the kinetics of single-vesicle catecholamine release from adrenal chromaffin cells stimulated with acetylcholine or high potassium (K(+)) have been recently found between normotensive Wistar rats (NWRs) and spontaneously hypertensive rats (SHRs). Such differences could be explained on the basis of distinct mechanisms of calcium (Ca(2+)) handling by chromaffin cells of NWRs and SHRs. We have explored here this hypothesis in adrenal medullary slices loaded with calcium fluorescent probes to measure the changes in Ca(2+) concentration in the cytosol ([Ca(2+)](c)), endoplasmic reticulum ([Ca(2+)](er)), and mitochondria ([Ca(2+)](m)). We found the following differences on calcium handling in SHRs, as compared with NWR: (i) higher basal [Ca(2+)](c) and basal [Ca(2+)](m); (ii) greater [Ca(2+)](c) elevations elicited by acetylcholine and K(+), with faster activation but slower inactivation; (iii) greater [Ca(2+)](c) elevations elicited by CRT (a mixture of caffeine, ryanodine, and thapsigargin) and by the mitochondrial protonophore FCCP (carbonylcyanide p-(trifluoromethoxy) phenylhydrazone). The higher basal [Ca(2+)](c) and [Ca(2+)](m) suggest an enhanced mitochondrial Ca(2+) uptake, and the greater [Ca(2+)](c) elevations produced by FCCP indicates a higher mitochondrial Ca(2+) release into the cytosol. This alteration of intracellular Ca(2+) movements could explain the greater quantal catecholamine release responses seen in SHRs, as compared with NWRs in previous studies. Furthermore, enhanced mitochondrial Ca(2+) cycling may be the basis for the dysfunction of mitochondrial bioenergetics, reported to be present in hypertensive states.

Topics: Acetylcholine; Adrenal Medulla; Animals; Caffeine; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Chromaffin Cells; Cytosol; Endoplasmic Reticulum; Fura-2; Hypertension; In Vitro Techniques; Male; Mitochondria; Movement; Potassium; Rats; Rats, Inbred SHR; Ryanodine; Thapsigargin

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 micromol/L) or gadolinium (100 micromol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension.

Topics: Animals; Aorta; Blood Pressure; Body Weight; Calcium; Calcium Channels; Cytosol; Disease Models, Animal; Enzyme Inhibitors; Homeostasis; Hypertension; Male; Membrane Glycoproteins; Muscle Contraction; ORAI1 Protein; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Stromal Interaction Molecule 1; Thapsigargin

Here, we present the first study on the effects of compounds that interfere with calcium (Ca(2+)) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K(+) pulses triggered spike bursts of secretion by Ca(2+) entry through Ca(2+) channels. ER Ca(2+) release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K(+), CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca(2+) handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K(+) were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca(2+) entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.

Topics: Animals; Caffeine; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Signaling; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Catecholamines; Cell Separation; Chromaffin Cells; Endoplasmic Reticulum; Enzyme Inhibitors; Hypertension; Mitochondria; Potassium; Rats; Rats, Inbred SHR; Rats, Sprague-Dawley; Ryanodine; Thapsigargin; Uncoupling Agents

We recently showed that increased expression of the transient receptor potential canonical Type 3 (TRPC3) channel is associated with genetic hypertension. It is unknown whether store-operated TRPC3 channels, which are activated after depletion of intracellular stores, or second messenger-operated TRPC3 channels, which are activated by 1-oleoyl-2-acetyl-sn-glycerol, show augmented responses in monocytes in genetic hypertension and support the development of vascular disease.. Using the fluorescent-dye technique, we studied store-depleted and thapsigargin-induced, store-operated calcium influx and 1-oleoyl-2-acetyl-sn-glycerol-induced second messenger-operated calcium influx into monocytes from spontaneously hypertensive rats (SHRs) and from normotensive Wistar-Kyoto rats (WKYs). The RNA interference for the downregulation of TRPC3 in monocytes by small, interfering RNA (siRNA) was performed and evaluated using in-cell Western assay.. Thapsigargin-induced, store-operated calcium influx was significantly elevated in SHRs and was approximately double that observed in WKYs. In the presence of nimodipine, the thapsigargin-induced, store-operated calcium influx was also significantly higher in SHRs compared with WKYs. After stimulation of monocytes by angiotensin II, calcium influx was significantly elevated in SHRs, and was approximately double that observed in WKYs. The 1-oleoyl-2-acetyl-sn-glycerol-induced, second messenger-operated calcium influx was also significantly elevated in SHRs compared with WKYs. Thapsigargin-induced, store-operated calcium influx was reduced by the inhibitor 2-aminoethoxydiphenyl borane. After TRPC3 knockdown, the thapsigargin-induced, store-operated calcium influx, as well as 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx, was significantly more reduced in cells from SHRs compared with WKYs.. The increased store-operated and second messenger-operated calcium influx through TRPC3 channels in monocytes from SHRs may be responsible for a more aggressive effect in promoting vascular disease in genetic hypertension.

Topics: Animals; Calcium; Diglycerides; Down-Regulation; Enzyme Inhibitors; Hypertension; Male; Monocytes; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Small Interfering; Second Messenger Systems; Thapsigargin; TRPC Cation Channels

Hypertension leads to impaired contractile function. This study examined the impact of inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by thapsigargin or cyclopiazonic acid (CPA) on cardiac contractile function in ventricular myocytes from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Mechanical properties were examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ transients were evaluated as fura-2 fluorescent intensity (FFI), excitation-induced change in FFI (DeltaFFI = peak-basal), and fluorescence decay rate (tau). Expression of Ca2+ regulatory proteins SERCA2a, Na+-Ca2+ exchanger (NCX), and phospholamban (PLB) were assessed by reverse transcriptase polymerase chain reaction and Western blot. SHR rats exhibited elevated blood pressure. SHR myocytes displayed decreased PS +/- dL/dt, peak FFI, and DeltaFFI; shortened TPS; prolonged tau with normal TR90; and basal FFI compared with WKY myocytes. Inhibition of SERCA with thapsigargin (5 microM) or CPA (10 microM) significantly depressed PS +/- dL/dt, baseline FFI, and DeltaFFI, and prolonged TPS, TR90, and tau in WKY myocytes. However, SHR myocytes were relatively insensitive to thapsigargin or CPA with only TPS and TR90 prolonged. Both mRNA and protein expressions of NCX and PLB were significantly enhanced, whereas SERCA2a protein abundance was reduced in SHR rats compared with the WKY group. Our data suggest that inhibition of SERCA function differentially affected cardiac contractile function in ventricular myocytes from normotensive and hypertensive rats possibly through reduced SERCA2a, elevated PLB, and NCX expression under hypertension.

Topics: Animals; Calcium-Binding Proteins; Calcium-Transporting ATPases; Cells, Cultured; Enzyme Inhibitors; Gene Expression; Heart Ventricles; Hypertension; Indoles; Intracellular Fluid; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Rats; Rats, Inbred SHR; Thapsigargin

The present study examined the role of L-/T-type Ca channels and the interaction between these channels and protein kinase C (PKC) in hypertension. The isolated perfused hydronephrotic rat kidney model was used to visualize directly the renal microvascular effects of L-/T-type Ca channel blockers (nifedipine and mibefradil, respectively). Nifedipine reversed the angiotensin II-induced constriction of afferent, but not efferent, arterioles in kidneys from Wistar-Kyoto rats (WKY), and similar magnitude in dilation was observed in spontaneously hypertensive rats (SHR). Although mibefradil elicited dilation of both arterioles, the afferent arteriolar dilation was less in SHR than in WKY (57+/-5% vs. 80+/-4% reversal at 1 micrommol/L). The pretreatment with staurosporine did not alter the angiotensin II-induced afferent arteriolar constriction in WKY, but attenuated this response in SHR. Furthermore, staurosporine enhanced the nifedipine-induced afferent arteriolar dilation (62+/-3% vs. 50+/-3% reversal at 10 nmol/L), and restored the attenuated afferent arteriolar response to mibefradil in SHR. The pretreatment with thapsigargin (a blocker of IP3-mediated intracellular calcium release) prevented the angiotensin II-induced afferent arteriolar constriction in WKY, but caused a significant constriction of afferent arterioles in SHR and efferent arterioles in WKY and SHR; in this setting, mibefradil did not alter efferent arteriolar tone. In conclusion, although both L-type (nifedipine) and T-type Ca channel blockers (mibefradil) exerted potent vasodilation of rat renal microvessels, these actions were modified by PKC, which determined the afferent arteriolar sensitivity to these blockers in SHR. Furthermore, the enhancement in nifedipine-induced afferent arteriolar dilation by staurosporine in SHR suggests that L-type Ca channel activity is augmented in hypertensive animals.

Topics: Angiotensin II; Animals; Arterioles; Calcium; Calcium Channel Blockers; Calcium Channels; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydronephrosis; Hypertension; Kidney; Male; Mibefradil; Microcirculation; Nifedipine; Perfusion; Protein Kinase C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Staurosporine; Thapsigargin; Vasodilator Agents

To investigate gender-dependent difference in the free cytosolic calcium concentration ([Ca2+ ]i ) response to angiotensin II (Ang II) in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). To further evaluate this gender-dependent difference by studying the role of thapsigargin-sensitive intracellular calcium stores and calcium influx in VSMC isolated from male and female SHR.. Confluent primary cultures of VSMC isolated from male (n = 14) and female (n = 14) SHR aged 10 weeks were used in this study. [Ca2+ ]i was measured by image analysis of single myocytes loaded with Fura-2. [Ca2+ ]i response of VSMC to Ang II was measured in the presence and absence of extracellular Ca2+, to evaluate the influence of Ca2+ influx. To characterize inositol triphosphate (IP3 )-sensitive sarcoplasmic reticulum calcium stores, thapsigargin-sensitive calcium stores were measured in VSMC isolated from SHR of both genders.. VSMC isolated from male SHR were characterized by an augmented [Ca2+ ]i response to angiotensin II in comparison with VSMC isolated from female SHR. Surprisingly, the thapsigargin-stimulated [Ca2+ ]i rise was found to be significantly greater in VSMC isolated from female SHR compared with VSMC isolated from male SHR. On the other hand, the gender-dependent difference in [Ca2+ ]i response to angiotensin II was abolished in the absence of extracellular calcium.. We demonstrated in VSMC isolated from SHR of both genders that a greater [Ca2+ ]i response to angiotensin II in male than female VSMC is dependent on Ca2+ influx.

Topics: Angiotensin II; Animals; Blood Pressure; Calcium; Enzyme Inhibitors; Extracellular Space; Female; Hypertension; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Sex Characteristics; Thapsigargin; Vasoconstrictor Agents

Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR.. Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process.. ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction.. We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.

Topics: Animals; Arteries; Calcium; Carotid Arteries; Charybdotoxin; Hypertension; In Vitro Techniques; Mesenteric Arteries; Peptides; Potassium Channels, Calcium-Activated; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reference Values; Rest; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin; Vasoconstriction

We examined mechanical alternans and electromechanical restitution in normal and failing rat hearts. Alternans occurred at 5 Hz in failing versus 9 Hz in control hearts and was reversed by 300 nM isoproterenol, 6 mM extracellular Ca(2+), 300 nM -BAY K 8644, or 50 nM ryanodine. Restitution curves comprised phase I, which was completed before relaxation of the steady-state beat, and phase II, which occurred later. Phase I action potential area and developed pressure ratios were significantly reduced in the failing versus control hearts. Phase II was a monoexponential increase in relative developed pressure as the extrasystolic interval was increased. The plateau of phase II was significantly elevated in failing hearts. Thapsigargin (3 microM) plus ryanodine (200 nM) potentiated phase I to a significantly greater extent in control versus failing hearts and abolished phase II in both groups. The results suggest that both regulation of Ca(2+) influx across the sarcolemma and Ca(2+) release by the sarcoplasmic reticulum may contribute to altered excitation-contraction coupling in the failing spontaneously hypertensive heart failure prone rat heart.

Topics: Action Potentials; Animals; Calcium; Heart Failure; Heart Rate; Hypertension; Isoproterenol; Male; Rats; Rats, Inbred BN; Rats, Inbred Strains; Rats, Inbred WF; Rats, Wistar; Ryanodine; Thapsigargin; Ventricular Dysfunction, Left; Ventricular Function, Left

To clarify whether the Ca2+ uptake function of the sarcoplasmic reticulum (SR) during arterial contraction is altered in hypertension, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit SR Ca2+-ATPase, on the contractile responses to Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of carotid arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of Bay k 8644 (1-300 nM) to the strips caused a concentration-dependent contraction that was larger in SHR than in WKY. The contractile responses to Bay k 8644 were augmented by CPA (10 microM) or thapsigargin (100 nM) in both strains. This augmentation was greater in SHR. Each of CPA and thapsigargin induced a relatively transient contraction, and both of these contractions were larger in SHR than in WKY. The basal 45Ca influx in this artery was larger in SHR than in WKY. The addition of caffeine (1-20 mM) caused a transient contraction that was larger in SHR than in WKY. Our results indicate that 1) the large Ca2+ influx during rest in the SHR carotid artery is strongly buffered by Ca2+ uptake into the superficial SR; and 2) the Ca2+ uptake function of the SR during the contraction with Bay k 8644 was increased in SHR compared with WKY. We conclude that the SHR carotid artery has an increased total capacity of SR for Ca2+ storage as an attempt to compensate for the large Ca2+ influx.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Buffers; Caffeine; Calcium; Calcium Channel Agonists; Carotid Arteries; Enzyme Inhibitors; Hypertension; In Vitro Techniques; Indoles; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin; Vasoconstriction

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adrenergic alpha-Agonists; Animals; Buffers; Calcium; Calcium Channel Agonists; Enzyme Inhibitors; Hypertension; In Vitro Techniques; Indoles; Mesenteric Arteries; Norepinephrine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Ryanodine; Sarcoplasmic Reticulum; Thapsigargin; Vasoconstriction

The interaction of the vascular smooth muscle cells (VSMCs) with the components of the matrix determines several functions of the cell, such as growth and differentiation. In contrast, an alteration in angiotensin (Ang) II-induced Ca(2+) mechanisms in VSMCs was reported in genetic hypertension. In this study, we wished to assess the effect of different components of the extracellular matrix on the increase of [Ca(2+)](i) induced by Ang II in VSMCs from spontaneously hypertensive rats (SHR) compared with those from normotensive Wistar-Kyoto rats (WKY). Results demonstrate for the first time that elements of the extracellular matrix modulate the Ang II-induced Ca(2+) transport mechanisms. This modulation is different in cells from WKY compared with those from SHR. Thus, growing cells from SHR on collagen I, collagen IV, fibronectin, vitronectin, or Matrigel induced a significant decrease in Ang II-induced Ca(2+) release from internal stores, whereas in cells from WKY, no effect could be observed except for those grown on collagen I, which increased Ca(2+) release. Fibronectin and vitronectin, however, induced a decrease in Ang II-induced Ca(2+) influx in WKY, whereas no effect could be observed in SHR. Conversely, collagen I and collagen IV induced an increase in this influx in SHR but not in WKY, whereas Matrigel increased the influx in both strains. These results suggest a modulation of the Ang II-associated signaling events by the matrix elements via the focal adhesion points. The understanding of these synergies should provide insight into issues such as development of hypertrophy of large vessels in hypertension.

Topics: Angiotensin II; Animals; Calcium; Cell Cycle; Cells, Cultured; Collagen; Cytoskeletal Proteins; Drug Combinations; Extracellular Matrix Proteins; Fibronectins; Hypertension; Laminin; Male; Muscle, Smooth, Vascular; Paxillin; Phosphoproteins; Proteoglycans; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin; Vitronectin

We have recently shown that insulin attenuates angiotensin II-induced intracellular Ca(2+) mobilization in human skin fibroblasts from normotensive subjects. This study was designed to investigate the effects of angiotensin II and the interactions between insulin and angiotensin II on intracellular Ca(2+) mobilization in skin fibroblasts from patients with essential hypertension. Fibroblasts were obtained from 9 normotensives and 18 hypertensives. Spectrofluorophotometric free Ca(2+) measurement was performed in monolayers of 24-hour serum-deprived cells. Resting intracellular Ca(2+) level and angiotensin II-stimulated intracellular Ca(2+) peak were higher in fibroblasts from hypertensives compared with those from normotensives. The effect of acute insulin exposure was evaluated in fibroblasts from hypertensives subdivided on the basis of insulin sensitivity. In insulin-sensitive hypertensives, insulin significantly blunted the effects of angiotensin II on intracellular Ca(2+) response, whereas in insulin-resistant patients, insulin did not modify intracellular Ca(2+) response to angiotensin II. Pertussis toxin, a G(ialpha)-inhibitor, reduced angiotensin II-stimulated Ca(2+) peak in insulin-sensitive but not in insulin-resistant hypertensives. In conclusion, the effects of angiotensin II on intracellular Ca(2+) mobilization are more pronounced in fibroblasts from hypertensives compared with those from normotensives, and the inhibitory effect of insulin is blunted in insulin-resistant hypertensives by a G(ialpha) pertussis toxin-sensitive abnormality.

Topics: Adult; Angiotensin II; Calcium; Cells, Cultured; Culture Media; Cytosol; Female; Fibroblasts; Humans; Hypertension; Insulin; Insulin Resistance; Male; Middle Aged; Pertussis Toxin; Skin; Thapsigargin; Virulence Factors, Bordetella

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Radioisotopes; Calcium-Transporting ATPases; Enzyme Inhibitors; Femoral Artery; Hypertension; Indoles; Male; Muscle Contraction; Nifedipine; Potassium; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Ryanodine; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Verapamil

We have examined the susceptibility to apoptosis in mesangial cells from spontaneously hypertensive rats (SHR) or from normotensive rats (WKY) and the possible involvement of nitric oxide in this process. Mesangial cells monolayers from either SHR or normal rats were incubated for 12 h in medium with or without fetal calf serum (FCS) and with or without thapsigargin (Tg, 10(-6) M). A series of cultures from rats of both groups was treated with N(G)-nitro-l-arginine methyl ester (l-NAME, 10(-4) M). We assessed apoptosis by propidium iodide staining, by TUNEL nitrite production (Griess reaction), by inducible nitric oxide synthase (iNOS) and Bcl-2 and Bax by Western blot. Incubated with a FCS-free medium, cells from SHR showed a significantly higher apoptotic rate (10.7 +/- 2.0) than with 10% FCS (10% FCS, 4.7 +/- 0.3), while WKY cells did not show this increment (10% FCS, 4.7 +/- 0.3; 0% FCS, 5.9 +/- 0. 3). Apoptosis in cells from WKY increased when incubated with thapsigargin in FCS-free medium (0% FCS+ Tg, 17.7 +/- 2.9%) and increased even more in SHR cells (0% FCS+ Tg, 19.7 +/- 2.9%). Treatment with l-NAME decreased thapsigargin-induced apoptosis in both SHR (8.2 +/- 2.4%) and WKY cells (9.3 +/- 2.4%). An increase in nitrite production and iNOS expression was detected in groups in which the apoptosis rate was elevated. A high rate of apoptosis was also associated with a decrease in the Bcl-2/Bax ratio. Our results indicate that in SHR cells, short-term serum deprivation and the increase in intracellular free calcium concentration with thapsigargin are able to enhance the apoptosis rate in primary cultures and that the expression of iNOS, and hence NO production, is involved in this effect.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Calcium; Cell Count; Cells, Cultured; Culture Media, Serum-Free; Enzyme Inhibitors; Glomerular Mesangium; Hypertension; In Situ Nick-End Labeling; Intracellular Fluid; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin

We studied the effect of basic fibroblast growth factor (b-FGF) on different Ca(2+) mechanisms elicited by angiotensin II (Ang II) in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Intracellular Ca(2+) (Ca(2+)(i)) variations were studied in cultured vascular smooth muscle cells (VSMCs) isolated from the aorta of 5- to 6-week-old WKY rats and SHR. Ca(2+)(i) was assessed in Fura-2-loaded cells with fluorescent imaging microscopy. Ang II subtype 1 receptor activation by Ang II (1 micromol/L) induced a transient increase in Ca(2+)(i) that was partially attenuated by genistein, a tyrosine kinase inhibitor. Pretreatment of VSMCs with b-FGF for 24 hours markedly stimulated the Ang II-induced Ca(2+)(i) release from the internal stores in WKY rats, whereas it was without effect in SHR. This was not consequent to a change in the affinity of Ang II subtype 1 receptors or an increase in their density. Inhibition of mitogen-activated protein kinase with PD 98059 reduced this stimulatory effect of the cytokine in the WKY rats. On the other hand, b-FGF stimulated the Ang II-induced Ca(2+) influx in both strains. Similar results were observed when Ca(2+) influx was induced with thapsigargin. Genistein and PD 98059 abolished the effect of b-FGF. These results show for the first time that b-FGF regulates Ca(2+) mechanisms induced by Ang II and that this regulation is different in SHR than in normotensive control animals. The extracellular signal-regulated kinase cascade is implicated in this cross-regulation with G protein-signaling pathway at 2 levels and possibly more: 1 at the tyrosine kinases and the other downstream of the extracellular signal-regulated kinase family. These results may prove useful in understanding the interaction between these 2 pathways and their implication in genetic hypertension.

Topics: Angiotensin II; Animals; Calcium; Calcium Signaling; Cell Size; DNA; Enzyme Inhibitors; Fibroblast Growth Factor 2; Flavonoids; Hypertension; In Vitro Techniques; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Protein Biosynthesis; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin

We studied the effect of propofol (5.6-560 micromol/L; 1-100 microg/mL) on the mechanisms involved in Ca(2+) mobilization elicited by angiotensin II (AngII) in Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We studied the variations in intracellular Ca(2+) ([Ca(2+)](i)) concentrations in cultured aortic vascular smooth muscle cells (VSMCs) isolated from 6-wk-old WKY and SHR rats loaded with the Ca(2+)-sensitive fluorescent dye, Fura-2, using fluorescent imaging microscopy. In the absence of external Ca(2+), AngII (1 micromol/L) induced a transient [Ca(2+)](i) mobilization from internal stores that was larger in SHR than in WKY rats. Ca(2+) influx was assessed after external Ca(2+) (1 mmol/L) reintroduction. Propofol (1-100 microg/mL) added 5 min before the experiments did not alter AngII-induced Ca(2+) release from internal stores in either strain. By contrast, Ca(2+) influx elicited by AngII was significantly decreased by propofol. This effect occurred at a smaller concentration of propofol in the SHR than in the WKY rats. When Ca(2+) stores were depleted by exposure of cells to thapsigargin, an inhibitor of the sarcoendoplasmic reticulum Ca(2+)-ATPase, reintroduction of Ca(2+) to the medium induced a capacitative Ca(2+) influx of similar magnitude than that elicited by AngII. This influx was also significantly decreased by propofol at 100 microg/mL ( WKY: 27 +/- 3% of control values, n = 107; SHR: 16 +/- 3%, n = 47; P < 0.001). In conclusion, propofol decreased AngII-induced Ca(2+) influx through voltage-independent channels, without altering Ca(2+) release from internal stores in aortic VSMCs. The hypertensive rats were found to be more sensitive to the effect of propofol than the normotensive rats. This suggests that the response of VSMCs to AngII may be altered by propofol.. In rat aortic vascular smooth muscle cells, propofol reduced angiotensin II-elicited Ca(2+) entry through capacitative Ca(2+) channels without altering Ca(2+) release from intracellular stores. Spontaneously hypertensive rats were more sensitive to these effects of propofol than normotensive rats. The response of vascular smooth muscle cells to angiotensin II may be altered by propofol.

Topics: Anesthetics, Intravenous; Angiotensin II; Animals; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Hypertension; Male; Muscle, Smooth, Vascular; Propofol; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin

To investigate the role of transforming growth factor-beta1 (TGF-beta1) on Ca2+-dependent mechanisms elicited by angiotensin II in aortic vascular smooth muscle cells (VSMC) of Wistar- Kyoto (WKY) rats and spontaneously hypertensive rats (SHR).. Cai2+ release induced by angiotensin II (1 micromol/ l) was studied in cultured VSMC isolated from the aortas of 6-week-old WKY rats and SHR. Intracellular Ca2+ (Cai2+) was assessed in Fura-2 loaded cells using fluorescent imaging microscopy. Angiotensin II receptors were analysed by binding studies.. Pretreatment of VSMC for 24 h with TGF-beta1 significantly increased angiotensin II-induced Cai2+ mobilization from internal stores in SHR, while Ca2+ influx was not altered. This effect involves tyrosine kinase and is not due to an increase in angiotensin II binding sites, or a change in the affinity of the receptors. By contrast, TGF-beta1 did not modify the response of VSMC from WKY rats to angiotensin II.. These results help our understanding of the interactions between the pathways activated by TGF-beta1 and the G protein-coupled receptor signalling pathway, and their role in genetic hypertension.

Topics: Angiotensin II; Animals; Binding Sites; Biological Transport; Calcium; Cells, Cultured; Enzyme Inhibitors; Hypertension; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction; Thapsigargin; Transforming Growth Factor beta

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.

Topics: Animals; Aorta, Thoracic; Bradykinin; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Enzyme Inhibitors; Hypertension; Intracellular Fluid; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin

We hypothesized that the cytoskeletal network in vascular smooth muscle cells (VSMC) is critical to the signaling pathways from angiotensin (ANG) II-receptor subtype 1 (AT(1)) activation to intracellular Ca(2+) (Ca(2+)(i)) release from internal stores and Ca(2+) influx. This was tested in spontaneously hypertensive rats (SHR), in which differences were reported in cultured aortic VSMC Ca(2+)(i) regulation and G protein function compared with those in normotensive Wistar-Kyoto (WKY) rats. In cultured aortic VSMC, disorganization of actin filaments with cytochalasin D (2 micromol/l) decreased the ANG II-induced Ca(2+)(i) release from internal stores and the ANG II-induced Ca(2+) influx in SHR in a reversible fashion, whereas it was without effect in WKY rats. On the other hand, blocking the dynamic state of the microtubule network significantly reduced ANG II-induced Ca(2+)(i) release from internal stores but was without effect on Ca(2+) influx in either SHR or WKY rats. This study demonstrates for the first time that, in the SHR, actin filaments play a major role in linking AT(1)-receptor activation to both Ca(2+)(i) release mechanisms and capacitative Ca(2+) influx. Furthermore, a functionally intact microtubule system is a necessary prerequisite for ANG II-induced Ca(2+)(i) release in both strains.

Topics: Actins; Angiotensin II; Animals; Calcium; Cells, Cultured; Enzyme Inhibitors; Hypertension; Male; Microtubules; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin

1. The effects of cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum (SR) Ca2+-ATPase, on twitch contraction and on the resting state of tension and intracellular Ca2+ level ([Ca2+]i) of the oesophageal striated muscle of stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY) were compared. 2. CPA (10 micronM) augmented the twitch contraction of oesophageal striated muscle preparations from both SHRSP and WKY, reducing the rate of relaxation (-dT/dt), and thus resulting in the prolongation of the time to 80% relaxation. The effect was significantly smaller in the SHRSP preparations. 3. In the resting state, CPA caused a sustained elevation of [Ca2+]i. The elevation was greater in the WKY preparations. Tension development accompanied by the elevation was observed in WKY preparations, but not in SHRSP preparations. 4. The sustained elevation of [Ca2+]i induced by CPA was eliminated by the removal of extracellular Ca2+. Both the elevated [Ca2+]i and tension in the preparations from WKY were reduced by flufenamic acid (100 micronM), mefenamic acid (100 micronM), lanthanum (La3+, 100 micronM), gadolinium (Gd3+, 100 micronM) and SK&F 96365 (100 micronM) but not by verapamil (10 micronM). 5. Thapsigargin (3 micronM), another SR Ca2+-ATPase inhibitor, produced similar effects on basal tension to those of CPA, although it reduced the amplitude of twitch contraction. 6. These results suggest that in the rat oesophageal striated muscle, (1) CPA extends the sequestrating time of Ca2+ into the SR, (2) CPA induces a Ca2+ influx mediated through verapamil-insensitive pathways, possibly nonselective cation channels, and (3) the mechanism of [Ca2+](i) modulation due to CPA-sensitive SR Ca2+-ATPase is deteriorated in the oesophageal striated muscle from SHRSP as compared with WKY preparations.

Topics: Animals; Blood Pressure; Body Weight; Calcium; Calcium-Transporting ATPases; Enzyme Inhibitors; Esophagus; Hypertension; In Vitro Techniques; Indoles; Ion Channels; Male; Muscle Contraction; Muscle, Smooth; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin

1. Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents which inhibit the Ca(2+)-ATPase of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. In resting strips preloaded with fura-PE3, the addition of CPA (10 microM) or thapsigargin (100 nM) caused an elevation of cytosolic Ca2+ level ([Ca2+]i) and a contraction. These responses were significantly greater in SHR than in WKY. 3. The additional of verapamil (3 microM) to the resting strips caused a decrease in resting [Ca2+]i, which was significantly greater in SHR than in WKY. In SHR, but not in WKY, this decrease was accompanied by a relaxation from the resting tone, suggesting the maintenance of myogenic tone in the SHR artery. 4. Verapamil (3 microM) abolished differences between SHR and WKY. The effects of verapamil were much greater on the contraction than on the [Ca2+]i. 5. The resting of Ca2+ influx in arteries measured after a 5 min incubation of the artery with 45Ca was not increased by CPA or thapsigargin in either SHR or WKY. The net Ca2+ entry measured after a 30 min incubation of the artery with 45Ca was decreased by CPA or thapsigargin in both SHR and WKY. The resting Ca2+ influx was significantly higher in SHR than in WKY, and was decreased by nifedipine (100 nM) in the SHR artery, but was unchanged in the WKY artery. 6. The resting 45Ca efflux from the artery was increased during the addition of CPA (10 microM). This increase was less in SHR than in WKY. The resting 45Ca efflux was the same in SHR and WKY. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels (VDCCs) was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional elimination of SR by CPA or thapsigargin caused a large elevation of [Ca2+]i and a potent contraction in this artery. During this process, the contraction was mainly due to the basal Ca2+ influx via L-type VDCCs. The present study also showed the existence of a relatively large compartment of [Ca2+]i which does not contribute to the co

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Radioisotopes; Enzyme Inhibitors; Femoral Artery; Hypertension; In Vitro Techniques; Indoles; Isometric Contraction; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Thapsigargin; Vasoconstriction; Vasodilator Agents; Verapamil

1. The full therapeutic potential of the main immunosuppressive drug, cyclosporin A (CsA), is limited because of its side effects, namely nephrotoxicity and hypertension. Several lines of evidence suggest that the origin of both side effects could be CsA-induced vasoconstriction. However, the underlying molecular mechanisms are not well understood. 2. Diameter measurements of rat isolated mesenteric arteries showed an increase in noradrenaline- and [Arg]8vasopressin-induced vasoconstriction when arteries were pretreated with CsA. 3. Measurements in cultured vascular smooth muscle cells (VSMC) of either cytosolic calcium concentration or of 45Ca2+ efflux showed that CsA potentiated the calcium influx to several vasoconstrictor hormones: [Arg]8vasopressin, angiotensin II, endothelin-1 and 5-hydroxytryptamine. On the other hand, 45Ca2+ efflux in response to thapsigargin, which depletes calcium from intracellular pools, was not potentiated by CsA. 45Ca2+ uptake was not altered by CsA or by any of the analogues tested. 4. Time-course studies in cultured VSMC showed that maximal CsA-induced Ca2+ potentiation occurred after ca. 20 h and this effect was reversed over approximately the next 20 h. 5. To investigate the possible role played by the known intracellular targets of CsA, namely cyclophilin and calcineurin, CsA derivatives with variable potencies with respect to their immunosuppressive activity, were tested on the calcium influx to [Arg]8vasopressin. Derivatives devoid of immunosuppressive activity (cyclosporin H, PSC-833) potentiated calcium signalling, while the potent immunosuppressant, FK520, a close derivative of FK506, and MeVal4CsA, an antagonist of the immunosuppressive effect of CsA did not. The latter compound was unable to reverse the calcium potentiating effect of CsA. 6. Our results show that CsA increases the calcium influx to vasoconstrictor hormones in smooth muscle cells, which presumably increases vasoconstriction. Loading of the intracellular calcium pools appears not to be involved. Experiments with derivatives of CsA and FK520 suggest that interactions with cyclophilins and calcineurin are not the mechanism involved. This indicates, for the first time, that the immunosuppressive activity can be dissociated from the calcium potentiating effect of CsA in vascular smooth muscle.

Topics: Adrenergic alpha-Agonists; Amino Acid Isomerases; Animals; Calcineurin; Calcium; Calmodulin-Binding Proteins; Carrier Proteins; Cyclosporine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Immunosuppressive Agents; Male; Mesenteric Arteries; Muscle, Smooth, Vascular; Norepinephrine; Peptidylprolyl Isomerase; Phosphoprotein Phosphatases; Rats; Rats, Inbred WKY; Thapsigargin; Vasoconstriction; Vasoconstrictor Agents; Vasopressins

This study addressed the controversy of whether endothelium-derived nitric oxide (NO) activity is increased or decreased in the hypertensive pulmonary vasculature of chronically hypoxic rats. Thapsigargin, a receptor-independent Ca2+ agonist and stimulator of endothelial NO production, was used to compare NO-mediated vasodilation in perfused lungs and conduit pulmonary artery rings isolated from adult male rats either kept at Denver's altitude of 5,280 ft (control pulmonary normotensive rats) or exposed for 4-5 wk to the simulated altitude of 17,000 ft (chronically hypoxic pulmonary hypertensive rats). Under baseline conditions, thapsigargin (10(-9)-10(-7) M) caused vasodilation in hypertensive lungs and vasoconstriction in normotensive lungs. Whereas the sustained vasodilation in hypertensive lungs was reversed to vasoconstriction by the inhibitor of NO synthase N(omega)-nitro-L-arginine (L-NNA; 10(-4) M), a transient vasodilation to thapsigargin in acutely vasoconstricted normotensive lungs was potentiated. As measured by a chemiluminescence assay, the recirculated perfusate of hypertensive lungs accumulated considerably higher levels of NO-containing compounds that did normotensive lungs, and thapsigargin-induced stimulation of NO-containing compounds accumulation was greater in hypertensive than in normotensive lungs. Similarly, low concentrations of thapsigargin (10(-10)-10(-9) M) caused greater endothelium-dependent L-NNA-reversible relaxation of hypertensive than of normotensive pulmonary artery rings. The increased sensitivity of hypertensive arteries to thapsigargin-induced relaxation was eliminated in nominally Ca(2+)-free medium and was not mimicked by ryanodine, a releaser of intracellular Ca2+. These results with thapsigargin, which acts on endothelial cells to stimulate Ca2+ influx and a sustained rise in intracellular Ca2+ concentration, support the idea that pulmonary vascular endothelium-derived NO activity is increased rather than decreased in chronic hypoxia-induced pulmonary hypertension in rats.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Hypoxia; Male; Nitric Oxide; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors

Strips of tail artery from stroke-prone spontaneously hypertensive rats (SHRSP), but not from normotensive Wistar Kyoto (WKY) rats, exhibit oscillatory activity after stimulation with norepinephrine. In addition, oscillatory activity is observed in response to tetraethylammonium (TEA) in vessels from both SHRSP and WKY rats. Mechanistically, the oscillatory contractions are associated with calcium (Ca2+)-driven action potentials. We have tested the hypothesis that intracellular Ca2+ stores participate in the generation of norepinephrine-induced oscillatory contractions in tail arteries from SHRSP. Additionally, the role of intracellular Ca2+ stores on TEA-induced contractions were evaluated. Contractile force in strips of tail artery from SHRSP and WKY rats was measured, using standard muscle bath procedures, and the effect of interventions that affect the storage of intracellular Ca2+ on the oscillatory contractions was evaluated. Depletion of intracellular Ca2+ stores, with ryanodine, or inhibition of Ca2+ uptake into the sarcoplasmic reticulum (SR), with thapsigargin and cyclopiazonic acid (CPA), did not inhibit oscillatory contractions induced by norepinephrine in SHRSP vessels. However, these agents inhibited the amplitude of TEA-induced contractions in WKY strips. Bay K 8644 and A23187 inhibited TEA-induced oscillatory contractions in WKY vessels. In SHRSP tail artery Bay K 8644 inhibited both norepinephrine and TEA-induced contractions, while A23187 did not have any effect. The phospholipase C inhibitor, NCDC (3X 10(-5) M), blocked oscillatory activity induced by norepinephrine in SHRSP tail artery and TEA-induced oscillations both in SHRSP and WKY vessels. These observations suggest that Ca2+ release and Ca2+ uptake into intracellular Ca2+ stores are not involved in the contraction-relaxation cycles that characterize norepinephrine-induced oscillatory activity in SHRSP tail artery. Similarly, SR Ca2+ stores may modulate but are not essential for TEA-induced oscillatory contractions.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Arteries; Blood Pressure; Calcimycin; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Channels, L-Type; Calcium-Transporting ATPases; Carbamates; Enzyme Inhibitors; Female; Hypertension; Indoles; Inositol 1,4,5-Trisphosphate Receptors; Ionophores; Male; Muscle, Smooth, Vascular; Periodicity; Phenylcarbamates; Phosphodiesterase Inhibitors; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cytoplasmic and Nuclear; Ryanodine; Sarcoplasmic Reticulum; Tail; Tetraethylammonium; Thapsigargin; Type C Phospholipases; Vasoconstriction

We examined contractile performance in perfused ventricles from normal rats and from SHHF/Mccfacp rats with end-stage heart failure. Changes in pacing frequency from 3 to 5 Hz evoked a complex response in normal rat myocardium. The first beat after a switch to 5 Hz was extremely weak, but each successive beat was stronger until force exceeded the 3 Hz steady state value by approximately 30%. Force then gradually declined to a new steady state where developed pressure was depressed but rate-pressure product was slightly greater than that at 3 Hz. By contrast, in failing SHHF/Mcc-facp hearts, an increase in pacing frequency from 3 to 5 Hz did not increase force development. Instead, the isovolumic left ventricles exhibited mechanical alternans. This alternation between weak and strong beats was abolished by 1 mM caffeine but restored by its washout. Inhibition of SR Ca2+ accumulation by 50-500 nM thapsigargin in normal ventricles did not evoke alternans when pacing frequencies were increased. The results indicate that mechanical alternans in failing rat hearts is due to altered reactions of the sarcoplasmic reticulum, but a decreased rate of Ca2+ accumulation is not the primary cause.

Topics: Animals; Calcium-Transporting ATPases; Cardiomyopathy, Dilated; Female; Heart; Hypertension; Male; Myocardial Contraction; Rats; Rats, Inbred WF; Rats, Mutant Strains; Rats, Sprague-Dawley; Reference Values; Species Specificity; Terpenes; Thapsigargin; Time Factors; Ventricular Pressure

To investigate whether the resting cytosolic free calcium concentration and the agonist-induced increase in this concentration in vascular endothelial cells from spontaneously hypertensive rats (SHR) differ from those in normotensive Wistar-Kyoto (WKY) rats.. The Fura-2 fluorescence technique was used to monitor changes in cytosolic free calcium concentration in isolated aortic endothelial cells from SHR (aged 12 weeks) and age-matched WKY rats.. Aortic endothelial cells from primary culture to the third passage were used. The cells were grown to a confluent monolayer on coverslips before the fluorescent measurement of cytosolic free calcium concentration. The resting cytosolic free calcium concentration and changes in it induced by bradykinin, endothelin 1, angiotensin II and thapsigargin were examined and compared between endothelial cells from SHR and WKY rats.. The resting cytosolic free calcium concentration in aortic endothelial cells was significantly lower in SHR than in WKY rats. Bradykinin induced a lesser increase in cytosolic free calcium concentration in endothelial cells from SHR than in those from WKY rats. However, aortic endothelial cells both from SHR and from WKY rats had a similar calcium response to endothelin 1 and angiotensin II. Furthermore, the thapsigargin-induced increase in cytosolic free calcium concentration was significantly less in SHR than in WKY rat endothelial cells. These results suggest that the altered calcium response to bradykinin in endothelial cells from SHR could be caused by an alteration of bradykinin receptors or changed bradykinin degradation. Alternatively, the intracellular calcium stores that are sensitive to thapsigargin and bradykinin may differ from those that are activated by endothelin 1 and angiotensin II in SHR aortic endothelial cells.. A lower level of resting cytosolic free calcium concentration and the impaired bradykinin-induced increases in it in SHR endothelial cells could reflect dysfunction of vascular endothelium underlying a reduced endothelium-dependent vasorelaxation in this genetic hypertension model.

Topics: Animals; Aorta; Bradykinin; Calcium; Cells, Cultured; Cytosol; Endothelins; Endothelium, Vascular; Enzyme Inhibitors; Hypertension; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Terpenes; Thapsigargin

Topics: Animals; Calcium-Transporting ATPases; Carotid Arteries; Cerebrovascular Disorders; Enzyme Inhibitors; Hypertension; Indoles; Male; Muscle Contraction; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Thapsigargin

To test the hypothesis that impaired Ca2+ recycling by the sarcoplasmic reticulum Ca-ATPase contributes to augmented force development in arteries from stroke-prone spontaneously hypertensive rats (SHRSP).. Force development to caffeine (0.3-30 mmol/l) in the absence of extracellular Ca2+ was compared in aortic strips from SHRSP and Wistar-Kyoto (WKY) rats. In another protocol the strips were rinsed at the peak of contraction to caffeine (20 mmol/l) and subsequently restimulated with the alkaloid. The second response, dependent on recycled Ca2+, was used as a measure of sarcoplasmic reticulum function. A third protocol evaluated caffeine-induced contractions after Ca2+ depletion and reloading. In these latter experiments the effects of thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, and ryanodine, an activator of sarcoplasmic reticulum Ca2+ release channels, were used to evaluate Ca2+ buffering. Finally, unidirectional 45Ca2+ influx was measured.. Contractions to caffeine (0.3-30 mmol/l) were larger in SHRSP aortic strips than in WKY rat strips. After a rinse at the peak of the initial response to caffeine, SHRSP segments contracted more when challenged a second time. Thapsigargin (0.3-10 mumol/l) caused a concentration-dependent contraction during Ca2+ loading that was greater in SHRSP than in WKY rat strips, and a concentration-dependent inhibition of caffeine-induced contraction with similar median inhibitory concentrations in the two groups. Ryanodine did not cause contraction during Ca2+ loading, but caffeine-induced contractions were reduced after ryanodine treatment in both groups. 45Ca2+ influx was increased in SHRSP aortic segments.. The greater force development to caffeine in SHRSP aortic strips probably reflects a greater storage of activator Ca2+ in the sarcoplasmic reticulum. On the basis of the pharmacological properties of thapsigargin and ryanodine, it appears that the larger store is caused by enhanced Ca2+ influx across the sarcolemma rather than by recycling of Ca2+ by sarcoplasmic reticulum Ca-ATPase. Experiments evaluating the secondary response to caffeine also support the interpretation that recycling of activator Ca2+ into the sarcoplasmic reticulum does not explain the augmented force development in SHRSP aortic segments.

Topics: Animals; Caffeine; Calcium; Dose-Response Relationship, Drug; Hypertension; In Vitro Techniques; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Ryanodine; Terpenes; Thapsigargin; Vasoconstriction

To evaluate the influence of the sarcoplasmic Ca(2+)-ATPase, isometric vasoconstrictions of aortic strips from spontaneously hypertensive rats from the Münster strain (SHR) and normotensive Wistar-Kyoto rats (WKY) were measured after inhibition of Ca(2+)-ATPase by thapsigargin. Inhibition of Ca(2+)-ATPase by thapsigargin caused a biphasic contractile response of the aorta in both SHR and WKY (maximum increase of tension: 1.7 +/- 0.3 x 10(-3) Newton and 2.1 +/- 0.3 x 10(-3) Newton, respectively; mean +/- SE). The second peak of the contractile response was abolished in the absence of external calcium or by inhibition of transplasmamembrane calcium influx by nifedipine, indicating that the second peak occurs as a consequence of calcium influx from the extracellular space. The initial peak of the contractile response after thapsigargin administration was abolished in the presence of an intracellular calcium antagonist, 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the initial response was due to calcium release from intracellular stores. Measurements using the fluorescent dye fura2 showed that thapsigargin increased the cytosolic free calcium concentration ([Ca2+]i) in SHR by 72.6 +/- 7.3 nmol/l (n = 34) and in WKY by 53.3 +/- 6.6 nmol/l (n = 39), showing no significant differences between the two strains. The inhibition of Ca(2+)-ATPase increases [Ca2+]i and causes vasoconstriction. The vasoconstriction produced by thapsigargin is not significantly different between SHR and WKY.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Cytosol; Gallic Acid; Hypertension; Muscle, Smooth; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Sarcoplasmic Reticulum; Terpenes; Thapsigargin; Vasoconstriction

To evaluate whether the distribution of intracellular free calcium may be impaired in primary hypertension.. Cytosolic free calcium and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR).. The concentrations of intracellular and stored calcium were investigated in cultured vascular smooth muscle cells from spontaneously hypertensive rats aged 6 months from the Münster strain (SHR) and from age-matched normotensive Wistar-Kyoto (WKY) rats. Vascular smooth muscle cells were grown on coverslips, and fluorescence measurements of the intracellular calcium concentration were performed using fura-2. The different effects of thapsigargin, a selective Ca-ATPase inhibitor, and of angiotensin II (Ang II) on the calcium storage pools were investigated.. In the absence of external calcium thapsigargin produced a dose-dependent transient increase in the concentration of intracellular calcium in vascular smooth muscle cells. The thapsigargin-induced maximum peak increase in the concentration of intracellular calcium was not significantly different in SHR and WKY rats. After depletion of the thapsigargin-sensitive calcium pools the addition of 100 nmol/l Ang II produced a rise in the concentration of intracellular calcium in vascular smooth muscle cells from SHR and WKY rats. Using vascular smooth muscle cells from the SHR the Ang II-induced increase in the concentration of intracellular calcium was not significantly different in the presence and absence of thapsigargin, indicating that the calcium pools depleted by thapsigargin and Ang II do not overlap significantly in vascular smooth muscle cells from SHR. In contrast, in the WKY rats the response to Ang II was significantly diminished after depletion of the thapsigargin-sensitive pool. When Ang II and thapsigargin were administered in the reverse order, i.e. Ang II before thapsigargin, the thapsigargin response was diminished in the WKY rats but not in the SHR.. SHR differ from WKY rats in having vascular smooth muscle cells that contain thapsigargin-sensitive calcium storage pools that are distinct from the Ang II-sensitive calcium pools.

Topics: Angiotensin II; Animals; Calcium; Calcium-Transporting ATPases; Cytosol; Hypertension; Intracellular Membranes; Male; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Reference Values; Terpenes; Thapsigargin

To study differences in cytosolic free calcium regulation between African Americans and Caucasians, using platelets as a model for studying cellular physiology.. Platelet calcium regulation in apparently healthy African American and Caucasian males was examined.. Using fura-2, calcium influx and cytosolic calcium extrusion were monitored after treatment with thapsigargin, an inhibitor of the Ca-ATPase in the dense tubular membrane system.. Compared with Caucasians, platelets from African Americans demonstrated a higher calcium influx into the cytosol (from the external medium and dense tubular membrane system), as well as enhanced calcium extrusion from the cytosol.. Such findings indicate that platelets from African Americans have increased intracellular calcium stores or increased cellular calcium turnover, or both. If these racial differences involve other cells, such as vascular smooth muscle cells, they could underlie the predisposition of African Americans to essential hypertension.

Topics: Adult; Black People; Blood Platelets; Calcium; Calcium-Transporting ATPases; Carrier Proteins; Cytosol; Egtazic Acid; Fura-2; Humans; Hypertension; In Vitro Techniques; Ion Transport; Lipids; Male; Sodium; Sodium-Calcium Exchanger; Terpenes; Thapsigargin; White People

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Cytosol; Hypertension; Isoquinolines; Muscle, Smooth, Vascular; Piperazines; Protein Kinase C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

Topics: Angiotensin II; Animals; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Glomerular Mesangium; Hypertension; Intracellular Fluid; Platelet-Derived Growth Factor; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Terpenes; Thapsigargin