Laura Corr

Marked increases in calcitonin gene-related peptide-containing nerves in the developing rat following long-term sympathectomy with guanethidine

Changes in the innervation of the cardiovascular system, urinogenital tract and sympathetic and non-sympathetic ganglia have been examined following long-term sympathectomy. Patterns of innervation were investigated using histochemical and immunohistochemical techniques, while levels of noradrenaline and neuropeptides were measured by neurochemical assays. Large doses of guanethidine (50 mg/kg) were given daily for 3 weeks to 8-day-old rat pups, which were killed at 6 or 20 weeks of age. In both age groups noradrenergic nerves were severely depleted or absent, while in some regions dramatic increases of calcitonin gene-related peptide levels were demonstrated. This was revealed by an increase in the density of nerve fibres and in calcitonin gene-related peptide content (up to 18-fold), most notably in the right atrium and superior cervical ganglion. No changes in substance P- or vasoactive intestinal polypeptide-immunolabelled nerves were seen. Conversely, short-term sympathectomy by 6-hydroxy-dopamine treatment caused a depletion of noradrenaline which was not accompanied by an increase in the number or content of calcitonin gene-related peptide-immunolabelled nerves. The possibility that nerve growth factor is involved in the mechanism of hyperinnervation by calcitonin gene-related peptide-containing sensory nerves following long-term sympathectomy is discussed.

Analysis of P2-purinoceptor subtypes on the smooth muscle and endothelium of rabbit coronary artery

Summary: We studied purinoceptor subtypes mediating constriction and dilatation in isolated rabbit coronary arteries with and without endothelium by comparing the effects of adenosine, ATP and the ATP analogues, α,β-methylene ATP and 2-methylthio ATP. For contraction, the rank order of agonist potency was α,β-methylene ATP > 2-methylthio ATP ≫ ATP; adenosine did not produce contraction. Removal of the endothelium did not affect these responses, but they were abolished by previous desensitisation with α,β-methylene ATP. For relaxation, adenosine, ATP, and 2-methylthio ATP were equipotent, but α,β-methylene ATP had no vasodilator effect and caused further contraction of the preparations. A transient contraction often preceded the relaxations to ATP at higher concentrations. The dilator responses to adenosine, ATP, and 2-methylthio ATP were significantly reduced but not abolished in preparations denuded of endothelium; previous desensitisation with α,β-methylene ATP had no effect on the relaxant responses but abolished the initial transient contractions to ATP and 2-methylthio ATP. These results indicate the presence of vasodilator P1- and P2Y-purinoceptors on smooth muscle and, to a lesser extent, on endothelium and P2X-purinoceptors, mediating transient vasoconstriction on smooth muscle alone. The presence of smooth muscle P2Y-purinoceptors is unusual and has functional implications regarding the nature of the response of the coronary artery to ATP.

Vasodilator response of coronary smooth muscle to the sympathetic co‐transmitters noradrenaline and adenosine 5′‐triphosphate

1 Vasodilator and vasoconstrictor responses to noradrenaline (NA), adenosine and adenosine 5′‐triphosphate (ATP) were examined in isolated ring segments of the left anterior descending coronary artery of the rabbit in the absence of endothelium. 2 NA caused dose‐dependent relaxation of potassium‐constricted vessels in the absence of β‐adrenoceptor blockade, with a pD2 of 5.96 ±0.21. This was inhibited by 1 μm propranolol. Constrictor responses of vessels at baseline tension were only seen at concentrations greater than 1 mm, and reached a maximum of 6% of the contraction to 30 mm KCl. 3 ATP caused relaxation of the potassium‐constricted ring segments in a dose‐dependent manner, although a transient constriction often preceded the relaxation. Adenosine was equipotent with ATP in producing relaxation; this was significantly inhibited by the P1‐purinoceptor antagonist, 8‐phenyltheophylline (8‐PT). The responses to ATP were little affected by 8‐PT, indicating that ATP was not acting through breakdown to adenosine. At basal tone, ATP produced transient vasoconstriction only at concentrations greater than 0.1 mm, reaching a maximum of 38% of the contraction to 30 mm KCl. 4 We conclude that in the rabbit coronary artery both NA and ATP produce vasodilatation by a direct action on the smooth muscle.

Sympathetic and nonsympathetic neuropeptide Y-containing nerves in the rat myocardium and coronary arteries.

We have examined the neuropeptide Y-containing intrinsic nerves of the heart in young (6-week-old) and adult (4-month-old) rats to determine whether they project to the coronary arteries or are capable of doing so if the neuropeptide Y-containing extrinsic nerves are removed. Chronic treatment of neonates with guanethidine was used to permanently destroy the sympathetic nerves. In the young treated animals, 33-54% of the neuropeptide Y remained in the heart despite a 90-99% reduction in norepinephrine; these proportions did not change in the animals that were allowed to develop to adulthood. The level of neuropeptide Y in the right atrium of young animals was unexpectedly high (252 +/- 28.7 pmol/g) compared with adults (75.4 +/- 18.8 pmol/g). The coronary arteries in the control rats received a moderately dense supply of neuropeptide Y-containing nerves; after guanethidine, all neuropeptide Y-containing nerves innervating the large coronary arteries disappeared, but some were still seen in association with small resistance vessels. No compensatory proliferation of the intrinsic neuropeptide Y-containing neurons occurred in the adult sympathectomized animals, and the intrinsic nerves did not reinnervate the large coronary arteries. These results are discussed in relation to the clinical syndrome of coronary artery spasm.

Dual Control by Nerves and Endothelial Cells of Arterial Blood Flow in Atherosclerosis

(1) Changes in the responses of vessels taken from Watanabe Heritable Hyperlipidaemic (WHHL) rabbits during the development of atherosclerosis at 4, 6 and 12 months were compared with those of vessels from control New Zealand White (NZW) rabbits at equivalent ages. (2) In vessels with severe atherosclerotic lesions (e.g. thoracic aorta) endothelial-mediated vasodilatation is attenuated, while direct responses to vascular smooth muscle are little changed. (3) In most other vessels (including mesenteric, hepatic, ear, coronary and saphenous arteries), where over 90% of endothelial cells appeared normal, endothelial-mediated vasodilator responses to acetylcholine and substance P were enhanced by 12 months in WHHL rabbits, while direct muscle vasoconstrictor and vasodilator responses showed gradual decline. Control NZW rabbits showed decline in both endothelial-mediated and direct muscle responses during the same period. (4) The basilar artery from WHHL rabbits showed little morphological evidence of serious lesions in either endothelial cells or smooth muscle up to 12 months, although endothelium-mediated vasodilatation was enhanced. This suggests that morphological criteria for assessing atherosclerotic damage do not necessarily correlate with changes in function. (5) Diminished sympathetic nerve-mediated vasoconstriction was demonstrated in 12-month-old WHHL mesenteric and hepatic arteries, although there was no reduction in adrenoceptor-mediated muscle responses. (6) It is suggested that the enhanced endothelial-mediated responses in these vessels may explain the well-known ‘compensatory vasodilatation’ characteristic of enlarged vessels in early atherosclerosis in which the lumen is not diminished and may even be enhanced. Speculations are made about the mechanism of enhanced endothelial-mediated vasodilatation. It is suggested that reduced nerve-mediated contraction may be an additional ‘trophic’ response contributing to ‘compensatory vasodilatation’ in early atherosclerosis. This study reinforces the view that different vessels respond differently in atherosclerosis and that there are marked variations with age and between the sexes in the changes occurring in the same vessel.

Neuropeptide Y in non-sympathetic nerves of the rat: Changes during maturation but not after guanethidine sympathectomy

Non-sympathetic neuropeptide Y-containing nerves were demonstrated by their persistence after destruction of sympathetic nerve terminals by acute 6-hydroxydopamine treatment for 48 h. In order to examine whether these neuropeptide Y-containing nerves reinnervate tissues following the loss of sympathetic nerves we administered guanethidine sulphate to one-week-old rat pups for three weeks to produce a complete and long-lasting sympathectomy and we monitored the innervation of the superior cervical ganglion, mesenteric vein, vas deferens and urinary bladder by noradrenaline- and neuropeptide Y-containing nerves two and 16 weeks later (assay and histochemical observations). By two weeks the reduction in neuropeptide Y content of tissues was similar to the reduction after acute sympathectomy with 6-hydroxydopamine treatment, indicating that there was no early reinnervation by non-sympathetic neuropeptide Y-containing nerve fibres at a time when sensory transmitters increase. Furthermore, there was no reinnervation by neuropeptide Y-containing nerve fibres by the time these sympathectomized animals had reached maturity, 16 weeks after cessation of treatment. Neuropeptide Y levels increased in the superior cervical ganglion with normal maturation but decreased in the prostatic end of the vas deferens. A non-sympathetic source of neuropeptide Y demonstrated in the immature rat vas deferens was no longer evident in the mature animal.

Effects of age and hyperlipidemia on rabbit coronary responses to neuropeptide Y and the interaction with norepinephrine

In coronary arteries from New Zealand White (NZW) rabbits up to 12 months of age, both direct vasoconstriction to neuropeptide Y (NPY) and inhibition of relaxation to norepinephrine (NE) by NPY were age dependent (p < 0.02 and p < 0.05, respectively); maximal relaxation to NE was unaffected. NPY had no significant effect on arteries from NZW rabbits at 4 months of age, while vessels from Watanabe Hereditable Hyperlipidaemic (WHHL) rabbits showed enhanced direct (p < 0.001) and indirect effects of NPY (p < 0.02). We conclude that the postsynaptic vasoconstrictor effects of NPY on the epicardial coronary artery increase with age and the presence of hyperlipidemia.

Responses of coronary arteries to neurotransmitters: changes with sexual maturity in the female rabbit.

Vasomotor responses of isolated coronary arteries to peptide and nonpeptide neurotransmitter agents changed with the development of sexual maturity in female New Zealand white rabbits aged 4–12 months. There was a significant reduction from 4− to 12-month-old animals in both the direct smooth muscle vasodilator responses to calcitonin gene-related peptide (p < 0.01) and vasoactive intestinal polypeptide (p < 0.001), and in the endothelium-mediated response to substance P (p < 0.005). Vasodilator responses to concentrations of acetylcholine (ACh) >0.1 μM were virtually absent in the 6-and 12-month-old animals. No change in maximal relaxation to noradrenaline was seen with maturation, although there was a small significant increase in potency. The contractile response of the smooth muscle to 30 mM KCl declined steadily as the animals matured, but the maximal contraction to ACh (p < 0.05), neuropeptide Y (p < 0.02), and serotonin (p < 0.05) increased significantly between 4 and 12 months of age. These results indicate that following sexual maturation in the female rabbit, the epicardial coronary artery shows a significant increase in maximum responses to vasoconstrictor neurotransmitter agents and, at the same time, a marked decline in responses to some vasodilator agents, both those acting directly on the smooth muscle and those acting via the endothelium.

Magnesium inhibits the responses to neuropeptide Y in the rabbit coronary artery

Neuropeptide Y (NPY) has been shown to cause direct vasoconstriction of coronary arteries in many species, including dogs, rabbits and man, which is selectively inhibited by calcium-channel blocking agents. Recently, NPY has also been reported to inhibit the relaxation to noradrenaline in isolated rabbit coronary arteries, but the composition of the Krebs solution described in this study indicated that it contained no magnesium. Since magnesium is known to be a physiological antagonist of calcium and to have a profound influence on the contraction of coronary vascular smooth muscle, we examined the importance of magnesium in modulating both the direct vasoconstrictor response to NPY and the NPY-induced inhibition of relaxation to noradrenaline, using ring preparations of rabbit circumflex coronary artery.