guanosine-diphosphate and 2--5--dideoxyadenosine

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.

Topics: Adenylyl Cyclases; Animals; Colforsin; Cyclic AMP; Dideoxyadenosine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Intercellular Signaling Peptides and Proteins; Isoenzymes; Male; Mice; Peptides; Receptors, Cell Surface; Signal Transduction; Sodium Fluoride; Sperm Capacitation; Spermatozoa; Thionucleotides; Wasp Venoms

Behavioral studies have shown that mechanical hyperalgesia induced by intradermal injection of prostaglandin E2 is blocked by inhibitors of the cAMP second messenger system. Similarly, injection of prostaglandin E2 also induces a decrease in mechanical threshold and an increase in the number of action potentials elicited by test stimuli in most C-fibre nociceptors. This change is called sensitization. To further evaluate the degree of correlation between primary afferent sensitization and mechanical hyperalgesia, we conducted a study to evaluate the effect of agents known to block the cAMP second messenger system and behavioral manifestations of mechanical hyperalgesia following injection of prostaglandin E2. The agents tested were guanosine 5'-O-(2-thiodiphosphate), an inhibitor of stimulatory guanine nucleotide-binding regulatory proteins; 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase; and Walsh Inhibitor Peptide, an inhibitor of cAMP-dependent protein kinase. Single fibre electrophysiologic studies of 138 C-fibres, innervating the dorsum of the hind paw, was done in male Sprague-Dawley rats. The number of spikes evoked by a 10 s application of a threshold von Frey hair were determined before and after intradermal injection of test agents administered alone and in combination with prostaglandin E2. Injection of prostaglandin E2 with the test agent vehicle (saline or distilled water) resulted in a significant decrease in von Frey hair threshold and an increase in the number of spikes generated in response to threshold von Frey hairs. In contrast, co-injection of prostaglandin E2 with guanosine-5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine or Walsh inhibitor peptide did not result in a significant decrease in von Frey hair mechanical threshold or increase in the number of spikes generated to the threshold stimuli, compared with vehicle/prostaglandin E2. It is suggested that guanosine 5'-O-(2-thiodiphosphate), 2',5'-dideoxyadenosine and Walsh inhibitor protein inhibited prostaglandin E2 sensitization of primary afferent C-fibres by inhibiting a stimulatory guanine nucleotide-binding regulatory protein, adenylyl cyclase, and protein kinase A, respectively. These results support the hypothesis that primary afferent sensitization by prostaglandin E2 underlies prostaglandin E2-induced hyperalgesia.

Topics: Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dideoxyadenosine; Dinoprostone; Enzyme Inhibitors; Evoked Potentials; Guanosine Diphosphate; Hyperalgesia; Intercellular Signaling Peptides and Proteins; Male; Mechanoreceptors; Nerve Fibers; Neurons, Afferent; Peptides; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Sensory Thresholds; Thionucleotides

Recent evidence strongly suggests that the hyperalgesia induced by agents acting directly on the primary afferent is mediated by stimulatory G-proteins and the cAMP second messenger system. In this study, we used the Randall-Selitto paw-pressure device to study hyperalgesia that develops in the streptozotocin-diabetic rat. Subcutaneous injection of streptozotocin in male Sprague-Dawley rats induced hyperglycemia and glucosuria detectable within 24 h of injection. A decrease in mechanical nociceptive threshold in the hindpaw was detected after one week. Intradermal injection of indomethacin, a cyclooxygenase inhibitor, had no significant effect on nociceptive threshold; and prostaglandin E2, which produces hyperalgesia by a direct action on the primary afferent, decreased nociceptive threshold similarly in streptozotocin-diabetic and control rats. Guanosine 5'-O-(2-thiodiphosphate), which blocks stimulatory G-proteins, attenuated the prostaglandin E2-hyperalgesia in both streptozotocin-diabetic and control rats, but had no effect on baseline nociceptive threshold in either group. Intradermal injection of either 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, or phosphodiesterase, which degrades cAMP, increased mechanical nociceptive threshold in streptozotocin-diabetic rats whilst not affecting mechanical nociceptive threshold in the control rats. Intradermal injection of 8-bromo cAMP, a membrane-permeable analog of cAMP, produced hyperalgesia of significantly greater magnitude in the streptozotocin-diabetic rats than the control rats. Intradermal injection of N6-cyclopentyl adenosine, an A1-type adenosine agonist, which can activate an inhibitory G-protein and decrease cAMP production, also increased nociceptive thresholds in streptozotocin-diabetic rats. This effect was blocked by pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenylate Cyclase Toxin; Animals; Diabetes Mellitus, Experimental; Dideoxyadenosine; Dinoprostone; GTP-Binding Proteins; Guanosine Diphosphate; Hyperalgesia; Indomethacin; Male; Mechanoreceptors; Pain; Pertussis Toxin; Rats; Rats, Sprague-Dawley; Sensory Thresholds; Thionucleotides; Virulence Factors, Bordetella