David P. Westfall

Contribution by purines to the neurogenic response of the vas deferens of the guinea pig

The specific purine-receptor antagonist, arylazido aminopropionyl ATP (ANAPP3), was used to determine if purines released from nerves of the guinea-pig was deferens contribute to the neurogenic response. ANAPP3, which is a photoaffinity label, antagonized contractile responses of the in vitro vas deferens to transmural stimulation, reversibly in the presence of the compound and irreversibly after its photoactivation in the presence of the tissues. The antagonism by ANAPP3 was augmented by depletion of norepinephrine produced by reserpine pretreatment. Responses of untreated tissues were only slightly antagonized by the alpha-adrenoceptor blocker prazosin. However, neurogenic responses were markedly reduced in the combined presence of ANAPP3 and prazosin. ANAPP3 did not affect the release of tritium from tissues prelabeled with [3H]norepinephrine. The initial phasic component of the neurogenic response was preferentially antagonized by ANAPP3 whereas the secondary more tonic component of the response was preferentially antagonized by prazosin and reserpine pretreatment. Small, residual responses remaining after chemical sympathectomy produced by 6-hydroxydopamine pretreatment were potentiated, rather than inhibited, by ANAPP3 and were, unlike untreated tissues, sensitive to atropine. These and previous findings indicate that ATP or a related purine, originating from adrenergic neurons, acts as a co-transmitter with norepinephrine in this tissue.

The postjunctional effects and neural release of purine compounds in the guinea-pig vas deferens☆

The smooth muscle of the in vitro guinea-pig vas deferens was shown to contract upon addition of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), the order of potency being ATP greater than ADP greater than AMP. Adenosine did not produce contraction. Pretreatment of animals with reserpine or treatment of tissues with an alpha-adrenoceptor blocking agent failed to alter the dose-response relationship for ATP. Because ATP is both a potent contractile agent and is present in the adrenergic storage complex, evidence was sought for the role of ATP as a possible co-transmitter following neural stimulation. Tissues preincubated in 3H-adenosine, a procedure which results in the incorporation of label into 3H-adenine nucleotides in the vas deferens, released significant amounts of tritium upon transmural stimulation. Because contraction per se can contribute to the tritium overflow, experiments were conducted with bathing solution made hypertonic with sucrose (12.5%). Hypertonic solution prevented the electrically induced tissue contraction, but failed to prevent a tetrodotoxin-sensitive release of tritium from tissue preincubated with either 3H-norepinephrine or 3H-adenosine. Because of the known association of ATP with norepinephrine in synaptic vesicles of adrenergic nerves and in view of the present evidence of a postjunctional action of ATP as well as the release of tritium from 3H-adenosine-treated vasa deferentia, it seems possible that in this tissue ATP, in addition to its other functions, may serve as a co-transmitter with norepinephrine.

High-pressure liquid chromatographic-fluorometric detection of adenosine and adenine nucleotides: application to endogenous content and electrically induced release of adenyl purines in guinea pig vas deferens.

To avoid some of the disadvantages associated with using radiolabeling to investigate adenyl purine content and release from excitable tissues, a reverse-phase high-pressure liquid chromatographic method utilizing fluorescence detection for the measurement of picomole amounts of endogenous ATP and its 6-amino purine analogs has been developed. This procedure has been used to determine the content of adenyl purines in the guinea pig vas deferens and that released from the tissue following stimulation of adrenergic nerves. The total tissue content was measured to be 1.6, 0.76, and 0.10 mumol/g of ATP, ADP, and AMP, respectively. However, adenosine could not be detected. Hypoxia caused a significant decrease in ATP content concomitant with an increase in adenosine content to 0.04 mumol/g. Following transmural electrical stimulation of the guniea pig vas deferens, the release of the following purine compounds was detected: ATP (0.106 nmol/g), ADP (0.242 nmol/g), AMP (0.035 nmol/g), and adenosine (0.454 nmol/g).

Evidence for a contribution by purines to the neurogenic response of the guinea-pig urinary bladder

In order to determine if ATP contributes as an excitatory transmitter in the guinea-pig bladder, experiments were conducted with ANAPP3, a photoaffinity analogue of ATP, which is an antagonist of adenine nucleotides in several other smooth muscles. With or without photoactivation with visible light, ANAPP3 antagonized contractile responses of in vitro strips of bladder to exogenous ATP. The antagonism was specific in that responses to acetylcholine and KCl were not affected by ANAPP3. Responses of strips of bladder to transmural electrical stimulation were not antagonized by ANAPP3 and were relatively insensitive to atropine. However, combined treatment with ANAPP3 and atropine produced a marked antagonism of the neurogenic response. In experiments with bladders obtained from animals pretreated with 6-hydroxydopamine, the ANAPP3-sensitive component of the neurogenic response was absent. These results suggest that acetylcholine, released from cholinergic nerves, and a purine, released from 6-hydroxy-dopamine-sensitive nerves, are both involved in motor transmission in this tissue.

Comparison of contractions of the smooth muscle of the guinea-pig vas deferens induced by ATP and related nucleotides.

The shape of contractile responses of the isolated guinea-pig vas deferens changes as ATP concentration is increased from 10(-7) to 10(-2) M. The ATP concentration-response curve is bimodal and reflects the change in response profile. Initially spike-like (10(-7) -3 x 10(-5) M) in nature, contractions acquire a secondary, slower tonic phase in transitional ATP concentrations (greater than or equal to 3 x 10(-5) M). At high ATP concentrations (10(-2) M) the secondary phase predominates. To determine if there are structural requirements for these complex effects, responses to ATP were compared to those elicited with analogs containing phosphate-chain, ribose and adenine modifications. In general, substitution of 5-anhydride linkages with methylene or imido bridges prolonged responses to low concentrations but at high concentrations both potentiated and abbreviated the responses. ATP gamma S, a substrate for phosphohydrolases which incorporate phosphate but which are less able to remove thiophosphate, produced responses with a greatly prolonged tonic phase. Removal of the 2-hydroxyl of ATP resulted in reduced potency at low concentrations while removal of the 3-hydroxyl was without effect. Modification of both the 1 and N6 positions of adenine substantially reduced agonist activity. Responses to ATP and the beta, gamma-methylene congener were unaffected by treatment with 10(-5) M indomethacin. The results indicate that more than one simple interaction of ATP with a receptor is involved in the production of responses to ATP. Of several hypotheses discussed, we favor one which suggests that the phasic responses to low concentrations of ATP are receptor-mediated and modified by tissue enzymes, while those to high concentrations are mediated, in part, by hydrolysis per se. The hypothesis, which realizes that conformational preferences may exist as well, is proposed for the vas deferens only.

Altered catecholamine contents in vascular and nonvascular tissues in genetically hypertensive rats.

We have measured the catecholamine (CA) contents in hearts, mesenteric vasculature, abdominal aorta, inferior vena cava, vasa deferentia and salivary glands from genetically hypertensive rats (SHR) and normotensive Kyoto-Wistar rats (WKY). We noted differences between the norepinephrine (NE) contents of individual tissues from SHR and WKY rats and have used two different analytical procedures for the measurement of NE to confirm these differences. Comparisons between tissue contents of NE in SHR and WKY rats indicated a greater content of NE in the following tissues from the SHR: heart, mesenteric artery, abdominal aorta, inferior vena cava and vasa deferentia. A modest elevation of NE [but not epinephrine (E)] was observed in adrenal glands from SHR rats. The NE contents of salivary glands from SHR and WKY rats were indistinguishable from each other. The results suggest that there may exist a generalized increase in NE contents in the peripheral vasculature of SHR rats. Furthermore, this increase is also present in the vasa deferentia, but not the salivary gland. The results draw attention to altered concentrations of NE in vascular and selected nonvascular tissues in the SHR.

A study of the atropine-resistant component of the neurogenic response of the rabbit urinary bladder

Rabbit bladder body was stimulated to contract by a number of agonists, of which bradykinin was the most potent, and ATP one of the least potent substances tested. The atropine-resistant component of the neurogenic response was unaffected by 2 X 10(-5) M chlorpheniramine or 10(-6) M methysergide, doses which suppressed responses to histamine or 5HT. Indomethacin 10(-5) M, or 10(-5) M capsaicin both reduced the atropine-resistant component. Following treatment with 10(-6) M atropine and 10(-5) M prazosin, 10(-4) M ANAPP3 produced a further suppression of the response, but did not antagonize the response to ATP. In the bladder body, the transmitter(s) responsible for the neurogenic response may be acetylcholine and prostaglandins and possibly ATP and substance P.

Comparison of the effects of arylazido aminopropionyl ATP (ANAPP3), an ATP antagonist, on responses of the smooth muscle of the guinea-pig vas deferens to ATP and related nucleotides

Abstract Contractile responses of the smooth muscle of the guinea-pig vas deferens to ATP and related analogs have been suggested previously to involve stimulation of cell-surface P2-purinergic receptors and obligatory hydrolysis, depending on the concentration and structure of the nucleotide. This hypothesis was further tested by evaluating the antagonistic effect of arylazido aminopropionyl ATP (ANAPP3), a specific P2-purinergic receptor antagonist, on concentration-response relationships and on the profiles of individual responses to ATP and analogs containing phosphate-chain, ribose and adenine modifications. The concentration-response curves for ATP, β, γ-methylene ATP (APPCP), β, γ-imido ATP (APPNP) and adenosine 5′-O-(3-thiotriphosphate) (ATPγS) were bimodal and suggested the existence of two receptors, one with high and one with low affinity for adenine nucleotides. The type of antagonism observed was nucleotide-specific, and concentration-response curves were monosigmoidal after ANAPP3 treatment. For ATP, antagonism was greatest at low concentrations ( 3 × 10−5 M) and resulted in a reduction in maximum response; for adenosine tetraphosphate (APPPP) the antagonism was pronounced at low and high concentrations; for ATPγS the antagonism was moderate at all concentrations. The concentration-response curves for a second group of less potent nucleotides was monosigmoidal. Included in this group was ADP, α, β-methylene ADP (APCP), 8-bromo ATP (8-BrATP) and 2-deoxy ATP (2d-ATP). Treatment with ANAPP3 shifted the concentration-response curves for these analogs to the right but did not reduce maximum responses. The characteristics of individual response profiles were structurally related. For most analogs responses to low concentrations of nucleotides were phasic and spike-like. Discernible tonic phases appeared with higher concentrations of ATP, ADP, ATPγS, APPPP and 2d-ATP. ANAPP3 antagonized the initial component of responses but did not affect the tonic phase. The initial phasic component of response has been related to receptor-mediated stimulation of the preparations, while the tonic component, characteristic of nucleotides without 5′-anhydride bridge substitutions, appears to be initiated by hydrolysis of the compounds, a process which is insensitive to ANAPP3.

Factors Influencing the Release of Purines and Norepinephrine in the Rabbit Portal Vein

Previous studies have shown that transmural electrical stimulation (TES) of the rabbit portal vein in vitro, results in the overflow of 3H-purines from tissues prelabelled with 3H-adenosine. The purpose of the present study was to assess the possible sites which contribute to the TES-induced overflow of purines in this adrenergically innervated tissue. The contribution to postjunctional elements to purine overflow was assessed with the alpha 1-adrenoceptor antagonist, prazosin. Prazosin (3 x 10(-7) M) did not affect the release of 3H-norepinephrine but markedly reduced the TES-induced contraction. The release of 3H-purines was reduced by 20% by prazosin, indicating that approximately 80% of the release is independent of the alpha 1-mediated postjunctional response and, therefore, probably originates from neuronal sites in the tissue. Two lines of evidence indicate that a considerable portion of the alpha 1-adrenoceptor-independent release of 3H-purines (i.e., in the presence of prazosin) arises from adrenergic nerves. First, the fractional release of 3H-purines was enhanced and reduced, respectively, by the alpha 2-adrenoceptor antagonist, yohimbine, and the alpha 2-adrenoceptor agonist, clonidine, in concentrations (10(-6) M) which did likewise to the fractional release of 3H-norepinephrine. Second, destruction of the adrenergic nerves by in vitro treatment with 6-hydroxydopamine reduced the fractional release of 3H-purines by 55%. The release of purines which remains after 6-hydroxydopamine treatment may occur from non-adrenergic nerves.

The effect of pretreatment with 6-hydroxydopamine on the norepinephrine concentration and sensitivity of the rat vas deferens.

Injection of 6-hydroxydopamine via the dorsal vein of the penis results in a marked depletion of the endogenous norepinephrine of the vas deferens. Seven days after pretreatment with 6-hydroxydopamine there is a shift to the left and increase in maxima of the dose-response curves for norepinephrine and methoxamine. The results indicate that pretreatment with 6-hydroxydopamine produces denervation of the vas deferens and that the in vitro tissue exhibits both prejunctional and postjunctional supersensitivity.