Selective blockade of angiotensin responses in the rabbit isolated vas deferens by angiotensin receptor antagonists

We examined the effect of two angiotensin receptor antagonists on neuromodulatory and prostaglandin-producing effects of angiotensin II in the rabbit isolated vas deferens because prior studies have established that angiotensins selectively influence the two neural events, one being adrenergic and the other nonadrenergic. Angiotensin II increased adrenergic neurotransmission and prostaglandin E synthesis in a concentration-dependent manner while depressing nonadrenergic neurotransmission. The [1-Sarcosine, 8-Alanine]-angiotensin II preferentially antagonized adrenergic neuromodulatory effects of angiotensin II. In contrast, the nonadrenergic neuromodulatory and prostaglandin E-releasing effects of angiotensin II were suppressed by [1-Sarcosine, 8-Alanine]-angiotensin II to a lesser extent. The nonpeptide angiotensin receptor antagonist, Dupont 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2(1)-(1-H-tetrazol-5-yl) biphenyl-4-yl)methyl] imidazole, potassium salt, exhibited the opposite selectivity. It eliminated the depression of nonadrenergic neurotransmission without significantly altering the potentiation of adrenergic neurotransmission caused by angiotensin II. The angiotensin-induced stimulation of prostaglandin E synthesis was also eliminated by this antagonist. These data suggest that angiotensin effects in the vas deferens are mediated by at least two types of angiotensin receptors.     

Adenosine receptor antagonism attenuates angiotensin II effects on adrenergic neurotransmission in the rabbit isolated vas deferens

Angiotensin II augments adrenergic neurotransmission in the rabbit isolated vas deferens and suppresses purinergic neurotransmission. This study tests the hypothesis that angiotensin II augments adrenergic neurotransmission by depressing the neuronal release of ATP, resulting in suppressed formation of the inhibitory neuromodulator, adenosine or a related purine. Exogenous ATP added to the vasa deferentia increased adenosine formation and depressed adrenergic neurotransmission thus providing indirect support for the hypothesis. The adenosine receptor antagonist, 8-(sulfophenyl)theophylline (10 and 100 microM) depressed responses to exogenous adenosine and ATP but did not alter contractile responses to nerve stimulation or exogenously administered norepinephrine thus indicating that endogenous adenosine had no basal influence upon neurotransmission. However, the 8-(sulfophenyl)theophylline reduced angiotensin II effects on both adrenergic neurogenic contractions and evoked norepinephrine release. Additionally, the augmentation of adrenergic neurogenic contractions by angiotensin II was enhanced in the presence of ATP. These results are consistent with an ATP involvement in angiotensin effects on adrenergic neurotransmission and contrary to the initial hypothesis, suggest that purines enhance adrenergic neurotransmission in the presence of angiotensin II.     

Alpha adrenergic receptors mediate angiotensin-induced prostaglandin production in the rabbit isolated vas deferens

The effect of alpha adrenergic receptor antagonists on concentration-dependent response to angiotensins II and III was examined in the electrically stimulated isolated rabbit vas deferens. The force generated by a nonadrenergic neural mechanism was reduced by both peptides whereas the force generated by adrenergic neural mechanisms was enhanced. Angiotensin III-induced inhibition of the nonadrenergic contraction was significantly greater than that of angiotensin II for all groups. Yohimbine (1 X 10(-4) M), an alpha-2 receptor antagonist, attenuated the depression of the nonadrenergic contraction produced by angiotensins II and III. Yohimbine (1 X 10(-5) and 1 X 10(-4) M) also significantly reduced angiotensin II-induced prostaglandin E (PGE) synthesis. Yohimbine only significantly altered the angiotensin III-induced PGE synthesis at an antagonist concentration of 1 X 10(-4) M. Rauwolscine (1 X 10(-8) and 1 X 10(-7) M) attenuated angiotensin II-induced PGE production and at a higher concentration (1 X 10(-6) M) reduced angiotensin III-induced PGE production. The alpha-1 antagonist, prazosin (1 X 10(-6) M), did not alter nonadrenergic contractile or PGE responses to either angiotensin. The alpha-2 agonist, clonidine, both inhibited the nonadrenergic neural contraction and enhanced PGE synthesis. We interpret these data to indicate that angiotensins II and III may act via separate mechanisms to induce PGE synthesis in the vas deferens, with angiotensin II effects being more dependent on norepinephrine release from adrenergic nerves.     

Neuromodulatory effect of the atrial natriuretic factor clearance receptor binding peptide, cANF(4-23)-NH2 in rabbit isolated vasa deferentia.

This study tests the hypothesis that atrial natriuretic factor (ANF) and the ANF clearance receptor binding peptide, cANF(4-23)-NH2 (cANF), inhibit adrenergic and purinergic neurotransmission in the rabbit isolated vas deferens by a pertussis toxin (PTX)-sensitive mechanism. The vas deferens is a unique model used in the study of autonomic neurotransmission inasmuch as it has both a purinergic or twitch contraction and an adrenergic or tonic contraction associated with its response to electrical stimulation. Both ANF and cANF (10(-11) to 10(-6) M) inhibited electrically induced purinergic and adrenergic contractile force generation in a concentration-dependent manner. The ANF effect on both purinergic and adrenergic contractions was blocked by PTX (100 ng/ml). The cANF effect on the adrenergic contraction was also PTX-sensitive. Both peptides also attenuated evoked norepinephrine release in a concentration-dependent manner by a PTX-sensitive mechanism. cANF (10(-7) M) had no effect on norepinephrine- or ATP-induced contractions as was shown previously for ANF (10(-7) M). Therefore, the inhibitory effects of ANF and cANF appear to be prejunctional, on the release of the neurotransmitters, norepinephrine and ATP, from the nerve terminal and not postjunctional on the smooth muscle. An effect of cANF on neurotransmission suggests that the reputed "silent" ANF clearance receptor has biological activity. PTX-sensitivity suggests the involvement of a guanine nucleotide-binding protein in mediating the neuromodulatory effect of atrial peptides.     

Adrenergic nerves mediate angiotensin-induced prostaglandin release in the rabbit vas deferens

The effect of sympathectomy and norepinephrine depletion on prostaglandin (PG) synthesis in response to angiotensins II and III was examined in isolated vasa deferentia of the rabbit. Either 6-hydroxydopamine or surgical denervation significantly depressed norepinephrine concentrations in the vas deferens relative to contralateral controls, thus confirming an effective sympathectomy. Guanethidine also resulted in a significant reduction in norepinephrine concentrations in the vas deferens. Base-line PGE production by the vasa deferentia was not altered by guanethidine but was attenuated by 6-hydroxydopamine and increased by surgical denervation. All the treatments reduced angiotensin-induced PGE synthesis. The effect of denervation on PGE synthesis was greater than that of norepinephrine depletion. We interpret these results to indicate that angiotensins stimulate PGE production by adrenergic nerves in the vas deferens and that released norepinephrine mediates a part of the PGE production in response to the angiotensins. The 6-hydroxydopamine experiments are consistent with the adrenergic nerves being the predominant source of PGE in this preparation.     

Atrial natriuretic factor inhibits adrenergic and purinergic neurotransmission in the rabbit isolated vas deferens

This study tests the hypothesis that atrial natriuretic factor (ANF) acts to inhibit neurotransmission in the rabbit vas deferens. The vas deferens is a unique model of autonomic neurotransmission in that it is composed of primarily nonvascular smooth muscle and has both a purinergic or twitch contraction and an adrenergic or phasic contraction associated with its response to electrical stimulation. In this study ANF was found to inhibit both adrenergic and purinergic neurotransmission in the rabbit vas deferens. ANF inhibited both the electrically induced phasic contraction and electrically induced norepinephrine release in a concentration-dependent manner over the ANF concentration range of 10(-10) to 10(-7) M. ANF at a concentration of 10(-7) M had no effect on norepinephrine-induced or ATP-induced contractions. Therefore, the neuromodulatory effect of ANF in the rabbit vas deferens appears to be prejunctional, on the release of the neurotransmitters norepinephrine and ATP from the nerve terminal and not postjunctional on the smooth muscle. Neither the alpha-2 antagonist rauwolscine nor the cyclooxygenase inhibitor indomethacin had any effect on the inhibitory effect of ANF on electrically induced twitch or phasic contractions. Additionally, ANF did not affect vasa deferentia prostaglandin E production. Therefore, the inhibitory neuromodulatory ANF effect is not mediated via alpha-2 adrenergic receptors or prostaglandin E production. The observed inhibitory neuromodulatory effects in this study may be involved in the hypotensive effects of ANF including natriuresis, diuresis and vasodilation.     

Pharmacological characterization of purinergic receptors in the rat vas deferens

Using the isolated rat vas deferens, we have confirmed the existence of P1 purinergic receptors whose activation results in an inhibition of the neurogenic twitch of the vas deferens. The observed order of potency for agonists (adenosine ethyl carboxamide greater than 2-chloroadenosine greater than adenosine greater than 5'-AMP greater than 5'-ADP greater than ATP) and antagonism of these effects by theophylline supports a P1-mediated response. Metabolically stable analogs of ATP elicited dose-dependent contractile responses which were quantitatively greater than, but qualitatively comparable to, ATP-induced responses. The order of potency for the eliciting contraction was the following: adenylyl-5-imidodiphosphate = beta-gamma-methylene ATP greater than adenosine tetraphosphate much greater than ATP greater than ADP. Interestingly, these compounds also produced an inhibition of the neurogenic twitch with a similar rank order of potency. This response was not due to the activation of P1 receptors insofar as high concentrations of theophylline failed to attenuate either the inhibition of the neurogenic twitch or the contractile response induced by these agonists. Thus, these data demonstrate the presence of both P1 and P2 purinergic receptors in the rat vas deferens. In addition, the data are consistent with the idea that two distinct classes of P2 receptors exist in this tissue. Furthermore, these data suggest that the rat vas deferens provides a useful tissue for studying compounds which interact with both major subtypes of purinergic receptors.     

Geographutoxin II, a novel peptide inhibitor of Na channels of skeletal muscles and autonomic nerves

Geographutoxin II (GTX II, 3 X 10(-9) to 10(-7) M) from a cone shell Conus geographus inhibited twitch responses of the isolated mouse diaphragm to direct stimulation in a dose-dependent manner. The contraction of the diaphragm induced by grayanotoxin I or veratridine was abolished by GTX II (3 X 10(-7) M), whereas the contractile response to KCI or caffeine was not affected. GTX II induced similar effects on isolated bullfrog sartorius muscles, but required higher concentrations (6 X 10(-7) to 3 X 10(-6) M). GTX II (greater than 10(-6) M) inhibited or abolished the action potential evoked in sartorius muscles markedly. In the isolated guinea pig vas deferens and ileum, GTX II caused a dose-dependent inhibition of the twitch responses to indirect nerve stimulation at concentrations of 3 X 10(-8) to 10(-6) M and 10(-7) to 10(-6) M, respectively. But the toxin had no effect on the dose contractile-response curves for norepinephrine, carbamylcholine or KCI in the vas deferens and for carbamylcholine or histamine in the ileum. GTX II (5 X 10(-8) to 10(-6) M) decreased norepinephrine release induced by veratridine from the vas deferens in a dose-dependent manner. These results suggest that GTX II blocks the voltage-sensitive Na channels in the cell membrane of skeletal muscles and autonomic nerves and these may play an important role in the mechanism of inhibitory effects of GTX II on contractile responses of these tissues to electrical stimulation.     

Purine nucleoside and nucleotide interactions on normal and subsensitive alpha adrenoreceptor responsiveness in guinea-pig vas deferens.

Adenosine and adenosine 5'-monophosphate (AMP) augment contractile responses to norepinephrine (NE) in isolated guinea-pig vas deferens. Dipyridamole slightly enhances, while theophylline antagonizes, adenosine effects on responses to NE. Adenosine triphosphate (ATP) and the nonhydrolyzable analog, adenosine 5'-(beta,gamma-imido)triphosphate (AppNp) depress responses to NE. Adenine and adenosine diphosphate (ADP) are ineffective in influencing alpha adrenoreceptor responsiveness. Repetitive stimulation of isolated vas deferens with maximal concentrations of NE markedly reduce the contractile response to test concentrations of 6 micron NE. Spontaneous resensitization of responses to NE to control levels occurs within 25 to 35 minutes after the end of desensitization treatment. Adenine nucleosides and nucleotides promote a more rapid rate of alpha adrenoreceptor resensitization, with a potency order: AMP greater than adenosine greater than ADP. Adenine and ATP did not influence the rate of alpha adrenoreceptor resensitization. The adenine nucleotides ADP, ATP and the analog AppNp elicit concentration-dependent contractions of guinea-pig vas deferens. Theophylline antagonizes this contractile activity to adenine nucleotides. AMP, adenosine and adenine are devoid of agonistic activity. In the presence of NE, however, AMP and adenosine produce contractile responses of isolated vas deferens strips, and the agonistic activity of ADP, ATP and AppNp is profoundly enhanced. Agonistic actions of purines in the presence of NE are antagonized by phentolamine much more effectively than by theophylline. The results suggest the existence of a purinergic receptor mediating excitatory responses of guinea-pig vas deferens. Furthermore, there appears to be mutual interaction between purinergic and alpha adrenoreceptor mechanisms. That adenyl derivatives are capable of augmenting subsensitive alpha adrenoreceptor responsiveness suggests that adenine nucleosides or nucleotides, released during sympathetic transmission, may be required for maintenance of normal alpha adrenoreceptor sensitivity.     

Cholera toxin and pertussis toxin stimulate prostaglandin E2 synthesis in a murine macrophage cell line

When RAW264.7 murine macrophages were incubated with cholera toxin or pertussis toxin, prostaglandin E2 (PGE2) synthesis was enhanced markedly. Cholera toxin and pertussis toxin added together synergistically stimulated PGE2 synthesis. Cholera toxin and pertussis toxin also stimulated cyclic AMP (cAMP) accumulation. However, PGE2 synthesis was independent of increases in cAMP, as neither forskolin nor isoproterenol, which increased cAMP accumulation, nor dibutyryl-cAMP had any effect on PGE2 synthesis. In intact cells, cholera toxin and pertussis toxin stimulated phospholipase A2 to enhance metabolism of phosphatidylinositol to lysophosphatidylinositol and glycerophosphoinositol, with time courses similar to their stimulation of PGE2 synthesis. Cholera toxin catalyzed ADP-ribosylation of proteins of Mr 45,000 and 49,000 in intact cells, whereas an additional substrate of Mr 41,000 was observed in vitro. Preincubation of intact cells with pertussis toxin blocked subsequent in vitro labeling of the Mr 41,000 protein by cholera toxin, suggesting that the same protein was ADP-ribosylated by both toxins. Western blot analysis using specific antisera against Gi, Go and Gs revealed that the Mr 41,000 substrate was bound by the anti-Gi and anti-Go but not anti-Gs. The present data suggest that guanine nucleotide binding regulatory proteins are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells. Furthermore, the possibility is raised that phospholipase A2 is regulated by both stimulatory and inhibitory guanine nucleotide binding proteins.     

Relationship between phospholipase C activation and prostaglandin E2 and cyclic adenosine monophosphate production in rabbit tubular epithelial cells. Effects of angiotensin, bradykinin, and arginine vasopressin.

By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+]i, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2+]i through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10(-9) to 10(-7) M stimulate cAMP indirectly through PGE2 production in distal cells. AII- and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in [Ca2+]i but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.     

Sympathetic denervation does not alter the density or properties of alpha-1 adrenergic receptors in rat vas deferens

Alpha-1 adrenergic receptors in surgically denervated rat vas deferens were studied using radioligand binding assays of [125I] BE 2254 ([125I]BE) and contraction measurements. Scatchard analysis of saturation isotherms of specific [125I]BE binding showed no change in the affinity or density of binding sites 4, 7 or 14 days after denervation of rat vas deferens. The potency of norepinephrine in inhibiting specific [125I]BE binding was also unchanged 7 days after denervation of vas deferens. The potency of phenylephrine in causing contraction in vitro did not change 4, 7 or 14 days after denervation of vas deferens; however, there was a significant increase in the maximum contractile response to phenylephrine at all time points. After partial inactivation of alpha-1 adrenergic receptors in vitro with phenoxybenzamine, there was an equivalent reduction in the number of [125I]BE binding sites in the control and 14-day denervated vas deferens. The equilibrium dissociation constants calculated from contractile measurements for norepinephrine were the same in the control and denervated tissues. However, there was a 2.2-fold increase in contractile sensitivity to norepinephrine 14 days after denervation and a 3.6-fold increase in contractile sensitivity to methacholine 7 days after denervation. These results show that surgical denervation of the rat vas deferens results in an increase in contractile sensitivity to norepinephrine and methacholine and an increase in maximum contraction. However, there is no change in alpha-1 adrenergic receptor density or properties at any time after denervation. Thus, alterations in alpha-1 adrenergic receptors do not contribute to contractile supersensitivity of denervated rat vas deferens.     

Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness

The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta2-adrenoceptor-mediated prejunctional facilitation and postjunctional inhibition of sympathetic neuroeffector transmission in the guinea pig vas deferens.

This study examines the role of prejunctional and postjunctional beta-adrenoceptors in the modulation of sympathetic cotransmission in the guinea pig vas deferens. The prejunctional involvement of beta-adrenoceptors was evaluated by testing the effects of several agonists and antagonists on the nerve stimulation-evoked overflow of ATP and norepinephrine (NE) from the "in vitro" vas deferens. The nonsubtype-selective beta-adrenoceptor agonist isoproterenol and the beta2-subtype-selective agonist clenbuterol increased, to a similar degree, the overflow of ATP and NE, while the beta1-subtype-selective agonist xamoterol and the beta3-subtype-selective agonist BRL 37 344 had no effect. Pretreatment with ICI 118, 551, a beta2-subtype-selective antagonist, abolished the facilitation of cotransmitter release by isoproterenol and clenbuterol, while the beta1-subtype-selective antagonist atenolol had no effect. Activation of beta-adrenoceptors by either isoproterenol or clenbuterol, but not by xamoterol and BRL 37 344, reduced the amplitude of contractions evoked by exogenously applied ATP. Pretreatment with propranolol or ICI 118, 551, but not atenolol, prevented these inhibitory effects. Isoproterenol in lower concentrations produced dose-dependent reduction of the purinergic but not the adrenergic phase of nerve stimulation-induced contraction of the guinea pig vas deferens. When applied in concentrations greater than 1 microM, isoproterenol, but not clenbuterol, actually produced a concentration-dependent facilitation of contractions evoked by both nerve stimulation and exogenously applied ATP. Antagonists of alpha-adrenoceptors blocked these facilitatory effects. Together, these results demonstrate that beta2-adrenoceptors can influence sympathetic neuroeffector transmission both prejunctionally, where they facilitate equally well the release of sympathetic cotransmitters and postjunctionally, where they inhibit smooth muscle contractions evoked by ATP.     

Angiotensin II-induced renal vasoconstriction in genetic hypertension.

Previous studies demonstrate that renovascular responses to angiotensin II (Ang II) are enhanced in spontaneously hypertensive rats (SHRs); however, it is possible that this hyperresponsiveness is mediated by Ang II-induced release of substances from the adrenal gland. Previous studies also show that pertussis toxin normalizes renovascular responses to Ang II in SHRs; however, it is possible that this response is mediated by effects of pertussis toxin on endogenous Ang II levels and/or the sympathoadrenal axis. The purpose of this study was 2-fold: 1) to determine whether the renovascular response to Ang II in SHRs is enhanced even in adrenalectomized SHRs and 2) to determine whether pertussis toxin normalizes enhanced renovascular responses to Ang II when pertussis toxin-induced changes in the renin-angiotensin system and the sympathoadrenal axis are prevented. SHRs and Wistar Kyoto (WKY) rats were anesthetized and administered 20 ml/kg 0.9% saline, and an infusion of aldosterone and hydrocortisone was initiated. After bilateral adrenalectomy, left renal denervation, and pretreatment with captopril, animals received an intrarenal artery infusion of Ang II at 10 ng/kg/min for 5 min. Ang II-induced changes in renal vascular resistance were greater in SHRs compared with WKY rats (p =. 010, n = 19/group). Pertussis toxin (10 microgram/kg i.v. 3 days before the experiment) attenuated Ang II-induced changes in renal vascular resistance in SHR (p <.05), but not in WKY rats (strain x treatment interaction: p =.046). These results suggest that the enhanced renovascular response to Ang II in SHRs is mediated by a G(i)-dependent pathway within the renal vasculature.     

Adenosine affects sympathetic neurotransmission at multiple sites in vivo

We examined the effects of adenosine and its analogs on sympathomimetic responses of pithed rats to electrical stimulation of preganglionic sympathetic nerves (ES) or to injections of nicotine, phenylephrine (PE) or isoproterenol (ISO). Four physiological indices of sympathetic neurotransmission were measured: blood pressure, heart rate and contractions of smooth muscle in vas deferens and eyelid. Elevation of arterial adenosine levels from 1.5 to 2 to 3 microM caused a 2- to 3-fold potentiation of nicotine-induced increases in blood pressure, heart rate and smooth muscle tension. Higher adenosine concentrations (3-4 microM) produced a smaller potentiation of the effects of nicotine. At 2 to 3 microM, adenosine had no effect on sympathomimetic responses to ES or PE. Higher concentrations (3-4 microM) attenuated pressor responses to ES and PE and the contractile responses of the vas deferens to ES; these levels also potentiated positive chronotropic responses to ISO. The adenosine analogs N-cyclopropylcarboxamido adenosine (N-CPCA), 2-chloroadenosine (2-CLA) and R- and S-phenylisopropyl adenosine (R-PIA and S-PIA) also reduced pressor responses to both ES and PE, with the potency order: N-CPCA greater than R-PIA greater than 2-CLA greater than S-PIA. These analogs exhibited this same potency series in attenuating contractile responses to ES in the vas deferens. However, all four analogs potentiated, at the lower doses tested, the contractile response of the vas deferens to PE; at higher concentrations, inhibition predominated. N-CPCA enhanced the chronotropic effects of ISO and ES.(ABSTRACT TRUNCATED AT 250 WORDS)     

The purinergic neurotransmitter revisited: A single substance or multiple players?

The past half century has witnessed tremendous advances in our understanding of extracellular purinergic signaling pathways. Purinergic neurotransmission, in particular, has emerged as a key contributor in the efficient control mechanisms in the nervous system. The identity of the purine neurotransmitter, however, remains controversial. Identifying it is difficult because purines are present in all cell types, have a large variety of cell sources, and are released via numerous pathways. Moreover, studies on purinergic neurotransmission have relied heavily on indirect measurements of integrated postjunctional responses that do not provide direct information for neurotransmitter identity. This paper discusses experimental support for adenosine 5′-triphosphate (ATP) as a neurotransmitter and recent evidence for possible contribution of other purines, in addition to or instead of ATP, in chemical neurotransmission in the peripheral, enteric and central nervous systems. Sites of release and action of purines in model systems such as vas deferens, blood vessels, urinary bladder and chromaffin cells are discussed. This is preceded by a brief discussion of studies demonstrating storage of purines in synaptic vesicles. We examine recent evidence for cell type targets (e.g., smooth muscle cells, interstitial cells, neurons and glia) for purine neurotransmitters in different systems. This is followed by brief discussion of mechanisms of terminating the action of purine neurotransmitters, including extracellular nucleotide hydrolysis and possible salvage and reuptake in the cell. The significance of direct neurotransmitter release measurements is highlighted. Possibilities for involvement of multiple purines (e.g., ATP, ADP, NAD⁺, ADP-ribose, adenosine, and diadenosine polyphosphates) in neurotransmission are considered throughout.     

Pravadoline: profile in isolated tissue preparations

Pravadoline is a novel cyclooxygenase-inhibiting analgesic with a preclinical profile of activity in vivo clearly distinct from that of other cyclooxygenase inhibitors. The purpose of the present study was to assess the possibility that pravadoline possesses pharmacologic actions in addition to inhibition of cyclooxygenase. The data demonstrate that pravadoline inhibits neuronally stimulated contractions of the guinea pig ileum and mouse vas deferens preparations. These actions of pravadoline are not shared by known cyclooxygenase inhibitors, but are mimicked by close structural analogs of pravadoline devoid of the ability to inhibit prostaglandin formation. Some structural analogs were inhibitory also in the rat vas deferens preparation. The inhibitory effects of pravadoline and a representative noncyclooxygenase-inhibiting analog in isolated tissue preparations are not mediated by an interaction with muscarinic cholinergic, adrenergic (alpha-1 or alpha-2), serotonin (5-hydroxytryptamine2 or 5-hydroxytryptamine3), opioid (mu, kappa or delta), purinergic (P1), neurokinin-1, bradykinin (B2) or prostaglandin (E2) receptors. It is concluded that pravadoline and aminoalkylinodole analogs are inhibitory in several bioassay systems via a presynaptic mechanism which does not involve inhibition of cyclooxygenase, or the activation or inhibition of several known receptors. The relevance of this mechanism to aminoalkyindole actions in vivo is the subject of future studies.     

Excitatory effect of the most potent marine toxin, maitotoxin, on the guinea-pig vas deferens

The most potent marine toxin, maitotoxin (MTX) (10(-9) to 3 X 10(-8) g/ml) caused a slower contraction of the isolated guinea-pig vas deferens (second component) in a dose-dependent manner after the first rapid phasic contraction (first component). The second component of the MTX-induced contraction was markedly inhibited by phentolamine and reserpine, whereas the first component remained unaffected. Both components were inhibited or abolished by verapamil or a Ca-free medium, but were not affected by atropine, chlorpheniramine or tetrodotoxin. The tissue Ca content of the vas deferens was increased by MTX 10(-9) to 3 X 10(-8) g/ml) in a dose-dependent manner. Furthermore, MTX (10(-9) to 3 X 10(-8) g/ml) caused a dose-dependent release of norepinephrine from the tissue, which was inhibited or abolished by verapamil or a Ca-free medium. In Na+-free medium, MTX still caused a profound increase in the tissue Ca content and a marked release of norepinephrine from the vas deferens. These results suggest that the major part of the first component is the result of a direct action of MTX on smooth muscle membrane, whereas the second component is primarily the result of indirect action mediated through the norepinephrine release from the adrenergic nerve terminals. It is also suggested that both components are possibly due to an increased Ca++ permeability of the voltage sensitive Ca++ channels in smooth muscle and nerve membrane.     

Potent excitatory effect of scaritoxin on the guinea-pig vas deferens, taenia caeci and ileum

Scaritoxin (STX), a potent toxic substance isolated from poisonous fish induced a dose-dependent contraction of the isolated guinea-pig vas deferens at concentrations of 10(-8) to 10(-6) g/ml. This contraction was abolished or inhibited by tetrodotoxin, low Na+ medium, phentolamine or reserpine, but not by atropine, chlorpheniramine or methysergide. STX shifted to the left the dose-contractile response curve for norepinephrine, KCI or acetylcholine in the vas deferens. STX caused a release of norepinephrine from the vas deferens, in a dose-dependent manner, which was blocked by tetrodotoxin, Mg++ or Ca++-free medium. In the guinea-pig taenia caeci or ileum, STX elicited a dose-dependent contraction. The contractions of both tissues were blocked completely by tetrodotoxin and were inhibited markedly by atropine but not by mecamylamine or chlorpheniramine. These results suggest that the STX-induced contraction is the result of an indirect action mediated through the norepinephrine release from adrenergic nerve terminals of the vas deferens and acetylcholine release from cholinergic nerve endings of the taenia caeci or the ileum. It is also suggested that STX causes an increase in Na+ permeability of the cell membrane of these tissues, which may play an important role in the release of chemical transmitters induced by STX.