Beta-adrenoceptor mediated facilitation of noradrenaline and adenosine 5'-triphosphate release from sympathetic nerves supplying the rat tail artery.

1. The effects of prejunctional beta-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5'-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro. 2. Isoprenaline (0.1 microM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train. 3. Isoprenaline (0.1 microM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps. 4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the beta-adrenoceptor antagonist, propranolol (0.1 microM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents. 5. Thus, activation of prejunctional beta-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.

Regional variations in the neural control of the female pig urethra.

OBJECTIVE: To establish the regional variation, if any, in the distribution of sympathetic and parasympathetic nerves within the urethra of the female pig and to correlate this with regional variations in the response of the smooth muscle to sympathetic and parasympathetic nerve stimulation and the application of phenylephrine and carbachol. MATERIALS AND METHODS: Female pig urethras were obtained from a local abattoir. Serial sections were cut from the proximal, middle and distal regions of the urethra and stained using tyrosine hydroxylase immunohistochemistry and acetylcholinesterase histochemistry for the demonstration of sympathetic and parasympathetic nerves, respectively. Strips of smooth muscle dissected from the same regions of the urethra were also mounted in organ baths to record isometric tension. Responses to nerve stimulation and alpha-adrenoceptor and muscarinic receptor activation were recorded. RESULTS: Tyrosine hydroxylase- and acetylcholinesterase-positive staining was demonstrated throughout the urethra. However, the density of sympathetic innervation was greatest in those strips dissected from the distal urethra, whilst the parasympathetic innervation was uniform throughout the length. Strips of urethral smooth muscle mounted for tension recording generated spontaneous tone. Smooth muscle dissected from the proximal urethra developed the greatest tone, whilst strips from the distal urethra generated significantly less. Responses to nerve stimulation were complex; sympathetic nerve stimulation elicited frequency-dependent contraction in all strips, but the response was most pronounced in the distal strips where tone was low. Conversely, parasympathetic nerve stimulation elicited the greatest contractile response from the proximal urethral strips. In all strips, but in particular those dissected from the proximal urethra, the contractile responses were attenuated by the occurrence of a non-adrenergic, non-cholinergic (NANC), non-nitrergic relaxation as stimulation frequency was increased. Phenylephrine and carbachol also produced concentration-dependent contraction of all urethral strips. Like the nerve-mediated responses, contraction in response to phenylephrine was most pronounced in the distal urethral strips whilst the response to carbachol was most pronounced in the proximal urethral strips. CONCLUSIONS: The results demonstrated a regional variation in the distribution of sympathetic nerves within the urethra of the female pig which would appear to be mirrored not only in the responsiveness of the tissue to sympathetic nerve stimulation but also in its response to alpha-adrenoceptor stimulation. In contrast, although no regional variation in the distribution of parasympathetic nerves could be demonstrated histologically, responses to nerve stimulation and the muscarinic agonist carbachol were most pronounced in the proximal urethral strips.

Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation.

1. Non-adrenergic, non-cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage-operated calcium channels (VOCCs). 2. EFS evoked frequency-dependent and tetrodotoxin-sensitive relaxations in the presence of propranolol (1 microM), phentolamine (1 microM) and scopolamine (1 microM). At low frequencies (< 12 Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long-lasting second phase. 3. Treatment with the NO-synthesis inhibitor NG-nitro-L-arginine (L-NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (> 12 Hz) only the first relaxation phase was affected. 4. Measurement of cyclic nucleotides in preparations subjected to continuous nerve-stimulation, revealed an increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels from 1.3 +/- 0.3 to 3.0 +/- 0.4 pmol mg-1 protein (P < 0.01). In the presence of L-NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5. Treatment with the N-type VOCC-inhibitor, omega-conotoxin GVIA (0.1 microM) reduced NO-dependent relaxations, the effect being most pronounced at low frequencies (1-4 Hz) of stimulation. The NO independent second phase of the relaxation, studied in the presence of L-NOARG (0.3 mM) at 16-30 Hz, was however markedly reduced or abolished by omega-conotoxin GVIA. omega-Conotoxin MVIIC (1 microM)or omega-agatoxin IVA (30 nM) had no effect on electrically evoked relaxations.6. These results suggest that NANC-nerve derived urethral relaxation in the pig consists of two apparently independent components. One is mediated by NO and associated with an increase in cyclic GMP content. The other mediator is unknown and produces relaxations not associated with changes in levels of cyclic nucleotides. The release of this mediator seems to involve the N-type VOCC, since the relaxation was markedly reduced or abolished by omega-conotoxin GVIA.

Characteristic features of inhibitory junction potentials evoked by single stimuli in the guinea-pig isolated taenia caeci.

1. Changes in membrane potential of the guinea-pig isolated taenia caeci evoked by single stimuli have been investigated using intracellular recording techniques. Nifedipine (10 microM) was used to arrest spontaneous muscle action potentials. Single stimuli elicited complex junction potentials which consisted of both excitatory and inhibitory components. 2. The excitatory component of the compound junction potential was unaffected by hexamethonium (100 microM) but abolished by atropine (1 microM) and omega-conotoxin GVIA (10-100 nM). 3. In the presence of atropine, single stimuli elicited fast inhibitory junction potentials (IJPs). IJPs were sometimes biphasic during repolarization with a noticeable 'slow tail'. Apamin (30-100 nM) potently inhibited the fast IJP and revealed an underlying slow IJP. 4. The fast IJP was also abolished by omega-conotoxin GVIA (100 nM). However, the slow IJP was insensitive to omega-conotoxin GVIA but was abolished by cadmium (30 microM). 5. Guanethidine (3 microM) and N omega-nitro-L-arginine (10-100 microM) had no detectable effects on either of the IJPs. The dye Reactive Blue 2 reduced the amplitude of the fast IJP but this reduction was associated with a membrane hyperpolarization. 6. The existence of two distinct IJPs in the guinea-pig taenia caeci has been demonstrated. The ability of omega-conotoxin GVIA to selectively abolish the fast IJP leaving the slow IJP intact suggests that separate nerves are involved in mediating these responses.

Regulation of tone in pig urethral smooth muscle.

Cystometry and urethral pressure profilometry performed in Large White pigs reveal a fall in urethral pressure before micturition. The area of highest pressure in the female is in the mid-urethra. Circular smooth muscle dissected from this region generates a high spontaneous tone, which is increased by phenylephrine and carbachol, but is unaffected by guanethidine and atropine. Electrical field stimulation using different parameters produces frequency-dependent relaxations or a combination of relaxation and contraction, all sensitive to tetrodotoxin. Only the contraction is inhibited by atropine and guanethidine. However, phasic relaxations are abolished by N-nitro-L-arginine, an effect partially reversed by L- but not by D-arginine. Oxyhemoglobin also marginally reduces the relaxations. Sodium nitroprusside produces concentration-dependent relaxations of the urethral smooth muscle, maximal at 10(-5) M. It is suggested that the urethral muscle generates a high myogenic tone that maintains continence and can be enhanced by adrenergic and cholinergic input, or inhibited by NANC innervation, partially mediated by the transmitter nitric oxide.