inositol-1-4-5-trisphosphate and Body-Weight

In this study we have investigated muscarinic M₁, M₃ receptor kinetics and the functional role of IP3 and cGMP in the corpus striatum of both young and old diabetic and insulin-treated diabetic rats.. Radioreceptor binding assays was done in the corpus striatum using specific antagonists QNB and DAMP. IP3 and cGMP assay using [3H]IP3 and [3H]cGMP Biotrak assay system kits.. M₁ receptor increased and M₃ receptor decreased in control old rats when compared with young control rats. In young diabetic groups M₁ receptor increased and M₃ receptor decreased. Old diabetic groups showed reversed M₁ and M₃ receptors compared with their controls. IP3 and cGMP content increased in old control rats compared with young control rats. IP3 content increased in young diabetic rats and decreased in old diabetic rats. cGMP content was increased significantly in both young and old diabetic groups. Insulin treatment reversed these altered parameters near to control.. Our studies showed that M₁ and M₃ receptors, IP3 and cGMP were functionally regulated during diabetes as function of age, which will have immense clinical significance.

Topics: Age Factors; Animals; Blood Glucose; Body Weight; Carrier Proteins; Corpus Striatum; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Inositol 1,4,5-Trisphosphate; Insulin; Intracellular Signaling Peptides and Proteins; Male; Rats; Rats, Wistar; Receptor, Muscarinic M1; Receptor, Muscarinic M3; Streptozocin

Ca2+ signaling and exocytosis are highly polarized functions of pancreatic acinar cells. The role of cellular architecture in these activities and the capacity of animals to tolerate aberrant acinar cell function are not known. A key regulator of acinar cell polarity is Mist1, a basic helix-loop-helix transcription factor. Ca2+ signaling and amylase release were examined in pancreatic acini of wild type and Mist1 null mice to gain insight into the importance of cellular architecture for Ca2+ signaling and regulated exocytosis. Mist1-/- acinar cells exhibited dramatically altered Ca2+ signaling with up-regulation of the cholecystokinin receptor but minimal effect upon expression of the M3 receptor. However, stimulation of inositol 1,4,5-trisphosphate production by cholecystokinin and carbachol was inefficient in Mist1-/- cells. Although agonist stimulation of Mist1-/- cells evoked a Ca2+ signal, often the Ca2+ increase was not in the form of typical Ca2+ oscillations but rather in the form of a peak/plateau-type response. Mist1-/- cells also displayed distorted apical-to-basal Ca2+ waves. The aberrant Ca2+ signaling was associated with mislocalization and reduced Ca2+ uptake by the mitochondria of stimulated Mist1-/- cells. Deletion of Mist1 also led to mislocalization of the Golgi apparatus and markedly reduced digestive enzyme content. The combination of aberrant Ca2+ signaling and reduced digestive enzyme content resulted in poor secretion of digestive enzymes. Yet, food consumption and growth of Mist1-/- mice were normal for at least 32 weeks. These findings reveal that Mist1 is critical to normal organelle localization in exocrine cells and highlight the critical importance of maintaining cellular architecture and polarized localization of cellular organelles in generating a propagating apical-to-basal Ca2+ wave. The studies also reveal the spare capacity of the exocrine pancreas that allows normal growth and development in the face of compromised exocrine pancreatic function.

Topics: Amylases; Animals; Basic Helix-Loop-Helix Transcription Factors; Body Weight; Calcium; Carbachol; Cholecystokinin; Cytosol; Dose-Response Relationship, Drug; Exocytosis; Feeding Behavior; Gene Deletion; Golgi Apparatus; Immunoblotting; Immunohistochemistry; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Mice, Transgenic; Mitochondria; Pancreas; Receptor, Muscarinic M3; Receptors, Cholecystokinin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Transcription Factors; Trypsin; Up-Regulation

alpha(1)-Adrenergic receptors (alpha(1A), alpha(1B), and alpha(1D)) are regulators of systemic arterial blood pressure and blood flow. Whereas vasoconstrictory action of the alpha(1A) and alpha(1D) subtypes is thought to be mainly responsible for this activity, the role of the alpha(1B)-adrenergic receptor (alpha(1B)AR) in this process is controversial. We have generated transgenic mice that overexpress either wild type or constitutively active alpha(1B)ARs. Transgenic expression was under the control of the isogenic promoter, thus assuring appropriate developmental and tissue-specific expression. Cardiovascular phenotypes displayed by transgenic mice included myocardial hypertrophy and hypotension. Indicative of cardiac hypertrophy, transgenic mice displayed an increased heart to body weight ratio, which was confirmed by the echocardiographic finding of an increased thickness of the interventricular septum and posterior wall. Functional deficits included an increased isovolumetric relaxation time, a decreased heart rate, and cardiac output. Transgenic mice were hypotensive and exhibited a decreased pressor response. Vasoconstrictory regulation by alpha(1B)AR was absent as shown by the lack of phenylephrine-induced contractile differences between ex vivo mesenteric artery preparations. Plasma epinephrine, norepinephrine, and cortisol levels were also reduced in transgenic mice, suggesting a loss of sympathetic nerve activity. Reduced catecholamine levels together with basal hypotension, bradycardia, reproductive problems, and weight loss suggest autonomic failure, a phenotype that is consistent with the multiple system atrophy-like neurodegeneration that has been reported previously in these mice. These results also suggest that this receptor subtype is not involved in the classic vasoconstrictory action of alpha(1)ARs that is important in systemic regulation of blood pressure.

Topics: Animals; Blood Pressure; Body Weight; Bradycardia; Cardiomegaly; Dose-Response Relationship, Drug; Echocardiography; Epinephrine; Femoral Artery; Heart Rate; Heart Septum; Humans; Hydrocortisone; Hypotension; Inositol 1,4,5-Trisphosphate; Kidney; Male; Mice; Mice, Knockout; Mice, Transgenic; Norepinephrine; Organ Culture Techniques; Organ Size; Phenotype; Phenylephrine; Promoter Regions, Genetic; Receptors, Adrenergic, alpha-1; Time Factors

Inositol 1,4,5-trisphosphate receptor (IP(3)-receptor) is a calcium channel, transporting calcium from intracellular stores to the cytoplasm. In kidney, IP(3)-receptors are involved in the signal transduction of various hormones. In our work we studied the effect of immobilization stress on the IP(3)-receptor's protein content in renal cortex and the medulla of normotensive and hypertensive rats. We detected both mRNA and type 1 IP(3)-receptor protein in medulla, but not in renal cortex. We found that this receptor was approximately twice as abundant in normotensive as in genetically hypertensive rat kidney. Immobilization stress decreased the amount of type 1 IP(3)-receptor in the renal medulla of normotensive rats approximately five times, while no effect due to single and/or repeated stress was observed in the renal medulla of spontaneously hypertensive rats. The results indicate that expression of type 1 IP(3)-receptor in renal medulla is modulated by hypertension and immobilization stress.

Topics: Alternative Splicing; Animals; Blood Pressure; Body Weight; Calcium Channels; Down-Regulation; Hypertension; Immobilization; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kidney Cortex; Kidney Medulla; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cytoplasmic and Nuclear; Stress, Physiological

Transgenic BDF-1 mice harboring an inducible, tissue-specific transgene for RNA antisense to Galphaq provide a model in which to study a loss-of-function mutant of Galphaq in vivo. Galphaq deficiency induced in liver and white adipose tissue at birth produced increased body mass and hyperadiposity within 5 weeks of birth that persisted throughout adult life. Galphaq-deficient adipocytes display reduced lipolytic responses, shown to reflect a newly discovered, alpha1-adrenergic regulation of lipolysis. This alpha1-adrenergic response via phosphoinositide hydrolysis and activation of protein kinase C is lacking in the Galphaq loss-of-function mutants in vivo and provides a basis for the increased fat accumulation.

Topics: Adipose Tissue; Animals; Body Weight; Cells, Cultured; Cyclic AMP; Diglycerides; Enzyme Activation; Female; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Lipolysis; Male; Mice; Mice, Transgenic; Phosphoenolpyruvate Carboxykinase (GTP); Protein Kinase C; RNA, Antisense; Type C Phospholipases

The action of deltamethrin, a potent type II synthetic pyrethroid insecticide, on the thymus of the Balb/c mouse was studied in vivo and in vitro. We found that deltamethrin produced atrophy in the thymus in a dose- and time-dependent fashion. The lowest effective dose was found to be 6 mg/kg, 24 hr after a single intraperitoneal treatment. Treated animals did not recover during the time-course of the experiment (365 days after treatment); however, deltamethrin did not affect the body weight of the treated animals during the course of the study. To determine if deltamethrin-induced [Ca2+]i signaling could lead to thymic atrophy via programmed cell death, mice were treated with 25 mg deltamethrin/kg for 24 hr or the isolated thymocyte suspension was treated with 50 microM deltamethrin. A significant stimulation of inositol 1,4,5-triphosphate (IP3) and inositol 1,4-diphosphate (IP2) production was found after 24 hr of deltamethrin-1R (active isomer) treatment. An inactive stereoisomer of deltamethrin (i.e. 1S) did not cause a significant rise in the production of 1P3 and 1P2. In addition, deltamethrin-1R induced a transient increase of [Ca2+]i mobilization in the thymocyte suspension after 10 min of in vitro treatment, and substantially reduced the rate of calcium-calmodulin (Ca/CaM)-dependent protein dephosphorylation in in vivo treated animals (25 mg deltamethrin/kg for 24 hr). The in vivo effects of deltamethrin treatment demonstrated induction of DNA fragmentation and cell death in thymocytes. Moreover, using a histochemical approach, it was evident that deltamethrin at 25 mg/kg was able to produce cell death in the thymus of treated animals 72 hr after treatment. In the present work, we found that cell death was apoptotic in nature as noted first by the inhibition of deltamethrin-induced cell death by aurintricarboxylic acid, an inhibitor of apoptosis, and second, by internucleosomal DNA fragmentation, a hallmark of apoptosis, produced by deltamethrin in treated animals as well in thymocyte suspensions. In addition, the involvement of the Ca/CaM-dependent protein phosphorylation-dephosphorylation cascade in the induction of apoptosis by deltamethrin was supported by the protective role of the calmodulin inhibitor trifluoperazine against the apoptotic effect of deltamethrin on thymocyte suspension. Our results suggest that deltamethrin induced thymus atrophy and altered the Ca/CaM-dependent protein kinase-phosphatase cascade, which might induce programm

Topics: Animals; Apoptosis; Atrophy; Body Weight; Cell Survival; Cells, Cultured; Concanavalin A; Dose-Response Relationship, Drug; Injections, Intraperitoneal; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Insecticides; Kinetics; Male; Mice; Mice, Inbred BALB C; Nitriles; Pyrethrins; Signal Transduction; Stereoisomerism; T-Lymphocytes; Thymus Gland

Because the phosphatidylinositol pathway may be part of the signaling system associated with stretch-induced release of atrial natriuretic peptide (ANP), we tested the hypothesis that formation of the intermediate inositol-1,4,5-trisphosphate (IP3) is impaired when ANP release is decreased in response to atrial stretch in hearts from aging genetically hypertensive (GH) rats. Immunoreactive ANP release into the coronary effluent and IP3 levels were studied in cardiac tissues of isolated perfused hearts from normotensive control (WAG) or GH rats aged 4, 11, or 16 months. Left atria were repeatedly distended and released with a latex balloon. ANP was measured in coronary effluent, and IP3 was measured in cardiac tissues. In all age groups, stretch and relief of stretch evoked considerably less ANP release in spontaneously beating hearts from GH than from WAG rats. Hearts from GH rats aged 16 months released no ANP, but electrical pacing restored some stretch-induced ANP secretion. With repeated stretch and release of stretch of the left atrium for 2 min, IP3 levels increased in left atrial tissue in WAG but not in GH hearts of all age groups. IP3 levels in (unstretched) left ventricles were much lower than in left atria and were unaltered by atrial stretch. In aging GH rats, the capacity to release ANP on atrial stretch is largely lost, in association with complete suppression of stimulus-induced increase in IP3 levels. These data support a role for IP3 in stretch-mediated atrial ANP secretion and suggest a progressive uncoupling of this signaling pathway in aging hypertensive rats.

Topics: Aging; Analysis of Variance; Angioplasty, Balloon; Animals; Atrial Function; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiac Pacing, Artificial; Compliance; Disease Models, Animal; Hypertension; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Myocardium; Organ Size; Rats; Rats, Wistar

Unilateral intraplantar injection of Freund's complete adjuvant (FCA) into 1 hind paw of rats was used as a model of peripheral inflammation and persistent pain in order to examine time course effects of a continuous barrage of nociceptive input on the second-messenger transducing systems in the spinal cord. cAMP, cGMP and inositol 1,4,5-trisphosphate (insP3) were extracted from the lumbosacral cord at days 1, 7, 14, 21 and 42 following FCA injection and quantified by either radioreceptor-assay (RRA) or radioimmunoassay (RIA). The lumbosacral contents of cAMP and cGMP when quantified in whole lumbosacral cord segment were not significantly changed by FCA treatment at all time points. InsP3 accumulation was significantly increased on days 14, 21 and 42 following FCA injection relative to sham-treated time-matched controls. However, cGMP and insP3 contents were significantly increased in the left longitudinal half of the lumbar enlargement ipsilateral to the injected paw on day 21 following FCA treatment, but not in the sham-treated time-matched controls. With [3H]insP3 as a ligand, Scatchard (Rosenthal) analyses of the concentration-dependent saturation curves showed that the densities (Bmax) of insP3 receptors (insP3R) were significantly increased throughout the time course of adjuvant-induced peripheral inflammation. The binding affinities (KD) for insP3R were significantly decreased on days 7, 14 and 21 following FCA injection corresponding to the times of most stable and peak inflammation. InsP3R from the cerebelli of the same rats as used in the lumbosacral insP3R characterization was used as a positive control in this study and did not show any change in both Bmax and KD as a result of FCA treatment, thus demonstrating that the changes in lumbosacral insP3R characteristics might be specific to the nociceptive sensory pathway such as the spinal cord. Thus it appears that sustained afferent nociceptive input induced by FCA injection increased the accumulation of cGMP, insP3 and insP3R density in the spinal cord through increased neuronal activities of functional receptors coupled to major classes of chemical mediators of nociception including neuropeptides and excitatory aminoacids. Changes in insP3 accumulation in the lumbosacral cord following FCA injection were significantly correlated with changes in insP3R density. Changes in the ratios of lumbosacral insP3 contents and insP3R density were also significantly correlated with changes in body weight

Topics: Animals; Body Weight; Cyclic AMP; Cyclic GMP; Freund's Adjuvant; Inflammation; Inositol 1,4,5-Trisphosphate; Kinetics; Male; Membranes; Nociceptors; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Spinal Cord; Thermodynamics

Chronic exposure of rats to low level (1 mg/kg. day) lead ingestion starting at a prenatal age reduced the number of IP3 receptors in ER and reduced the capacity of IP3 to increase Cai2+ in permeabilized neurons obtained from the cerebral cortices. Chronic exposure of adult rats to a comparable dose of lead did not cause these changes. A second stimulation of samples with IP3 in the absence of extracellular Ca2+ did not further increase Cai2+ suggesting the depletion of the IP3 sensitive Ca2+ pool. However, when IP4 preceded the second IP3 stimulation, an increase in Cai2+ was noted. In samples exposed to La3+ followed by IP3, Cai2+ remained elevated and did not return to the base line which suggests that the IP3 sensitive pool of Ca2+ is removed from neurons primarily by active extrusion. When IP4 was added to the samples exposed to La3+ and IP3, a significant decrease in Cai2+ was noted. These observations suggest that IP4 refills Ca2+ stores possibly by redistributing Ca2+ from cytoplasm to the stores. Chronic low level lead ingestion starting prenatally or at an adult age did not impair the effect of IP4 on Cai2+ suggesting that the IP4 induced redistribution of Ca2+ from cytoplasm to intracellular Ca2+ stores is not effected by chronic exposure of rats to low level lead ingestion. The present observations that chronic exposure of rats to low level lead ingestion does not effect the capacity of GTP to increase Cai2+ but attenuated the combined effects of GTP and IP3 in neurons further suggest that IP3 receptors are specifically down-regulated by prenatal lead exposure.

Topics: Animals; Body Weight; Calcium; Cell Membrane Permeability; Cerebral Cortex; Endoplasmic Reticulum; Female; Homeostasis; Inositol 1,4,5-Trisphosphate; Kinetics; Lead Poisoning; Neurons; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Time Factors