Postjunctional Vascular Alpha-2 Adrenoceptors: Modulation and Interactions

The work presented herein represents an examination of the alpha-adrenoceptors mediating contractions in isolated vascular preparations from the rabbit and the factors involved in modulating these responses. 1) The alpha-adrenoceptor population in the rabbit isolated lateral saphenous vein, based upon agonist and antagonist potency profiles, could not be ascribed to be either a homogeneous population of either postjunctional alpha1- or alpha2-adrenoceptors, but had characteristics of both. 2) A homogeneous population of postjunctional alpha2-adrenoceptors could be isolated in the lateral saphenous vein using receptor protection experiments with the combination of rauwolscine and phenoxybenzamine. 3) Only limited success was achieved in attempting to isolate a homogeneous population of postjunctional alpha1-adrenoceptors in the lateral saphenous vein using receptor protection experiments with the combination of YM 12617 and phenoxybenzamine. The residual response to NA remaining after this procedure had characteristics of a mixed population of both postjunctional alpha1- and alpha2-adrenoceptors. 4) A comparison of the effects of angiotensin II and Bay K 8644 revealed marked differences in their ability to modulate responses to NA mediated via postjunctional alpha1- and alpha2-adrenoceptors. AII produced a selective enhancement of responses mediated via postjunctional alpha2-adrenoceptors, while the action of Bay K 8644 was not dependent upon receptor subtype. 5) In the lateral saphenous vein after isolation of postjunctional alpha2-adrenoceptors, both Bay K 8644 enhanced responses to NA. The mechanism of this potentiation also appears to differ for these agents. Bay K 8644 enhanced responses mediated via voltage-dependent Ca2+ channels, while AII inhibited the influx of Ca2+ mediated via these channels. 6) The effects of A II on responses mediated via postjunctional alpha2-adrenoceptors, was mimicked by its physiological precursor angiotensin I, suggesting that local vascular production of A II may be important for the facilitatory action of this peptide. 7) Nifedipine, like Bay K 8644, had a non-differential effect on responses to NA mediated via postjunctional alpha1 and alpha2-adrenoceptors in a number of isolated vascular preparations. 8) Under normal experimental conditions, based upon agonist and antagonist potencies, the rabbit isolated distal saphenous artery contains a homogeneous population of postjunctional alpha1-adrenoceptors. 9) In the presence of A II, there was a marked increase in the responsiveness of the distal saphenous artery to UK-14304, which was prazosin-resistant, rauwolscine-sensitive, and so mediated via postjunctional alpha2-adrenoceptors. 10) After attempted isolation of postjunctional alpha2-adrenoceptors in the distal saphenous artery using receptor protection experiments, with the combination of rauwolscine and phenoxybenzamine, no responses were observed. 11) AII uncovered responses to alpha-adrenoceptor agonists after the combination of rauwolscine and phenoxybenzamine. The agonist and antagonist potencies after this protocol were consistent with a homogeneous population of postjunctional alpha2-adrenoceptors. 12) Some of the results in the present study indicate an interaction between postjunctional alpha1- and alpha2-adrenoceptors in vascular smooth muscle. The implications for such an interaction is discussed in detail. Furthermore, evidence is presented demonstrating an interaction between postjunctional alpha2-adrenoceptors and a number of vasoactive agents. 13) Sympathetic neurotransmission in the rabbit isolated distal saphenous artery is the resultant of an interaction between three receptor systems, postjunctional alpha1-and alpha2-adrenoceptors and purinoceptors. alpha1-adrenoceptors are of principal importance, although a role for the two other receptor systems can be demonstrated under the appropriate experimental conditions

Local neural and endothelial-mediated control of the rabbit central ear artery: Changes with chronic nerve stimulation, X-radiation and acrylamide poisoning

This thesis examines the neural and endothelium-mediated control of the rabbit isolated central ear artery. The preparation was also used as a model to investigate the plasticity of vascular sympathetic transmission following long term stimulation in vivo, and the pathological changes produced in local control mechanisms following x-irradiation and acrylamide poisoning. Calcitonin gene-related peptide- and substance P-like immunoreactive perivascular nerve fibres were localised and shown to be of sensory origin. Exogenous calcitonin gene-related peptide exhibited endothelium-independent relaxant responses and inhibitory postjunctional actions, but no prejunctional neuromodulation on the release of noradrenaline. Somatostatin-like immunoreactivity was also localised in perivascular nerves, and exogenous somatostatin had inhibitory neuromodulatory actions both pre- and postjunctionally in the rabbit ear artery. Neuropeptide Y appears to be colocalised with noradrenaline and adenosine 5'-triphosphate in sympathetic nerves in the rabbit ear artery. Exogenous neuropeptide Y inhibited the release of noradrenaline prejunctionally, as well as potentiated contractile responses postjunctionally. Plasticity of local control mechanisms in the rabbit ear artery was examined following chronic cold exposure, which is known to increase activity in sympathetic nerves. Preparations taken from young adult and aged cold-stressed animals showed that there was an age-related difference in the ability of the sympathetic transmission to adapt to chronic cold stress. Up to six weeks after exposure to a single dose of x-radiation, damage to perivascular nerves, endothelial cells, smooth muscle cells and fibroblasts were observed in the rabbit ear artery. The tissue content of noradrenaline was reduced, but neuropeptide Y and calcitonin gene-related peptide levels were unaltered. X-irradiation-induced changes in local neural, endothelium-mediated and direct smooth muscle responses in the rabbit ear artery are discussed. Acrylamide poisoning did not alter the tissue content of noradrenaline, neuropeptide Y or calcitonin gene-related peptide in the rabbit ear artery. Although actions mediated by the peptides examined were unaffected, sympathetic cotransmission as well as endothelium-dependent relaxant responses were attenuated

Pharmacology of the ovine uterine artery and umbilical blood vessels: Serotonergic and α-adrenergic mechanisms

Uteroplacental blood flow is critical for normal fetal growth and development. Uterine and umbilical vessels are under the influence of many factors which could have an impact on fetal well-being. Norepinephrine and 5-hydroxytryptamine are potent constrictors of the uterine and umbilical blood vessels. Isolated ovine uterine artery and umbilical blood vessels were used to study serotonergic and adrenergic mechanisms. The major findings were: (1) 5-hydroxytryptamine induced the hydrolysis of phosphoinositide via activation of 5-HT2 receptors in the ovine umbilical artery; (2) norepinephrine amplified 5-hydroxytryptamine-induced contraction of the ovine uterine artery via activation of [alpha]1-adrenoceptors; (3) [alpha]1-adrenoceptors were heterogenous in the ovine uterine artery and umbilical vein; and (4) activation of protein kinase C was involved in the contractions induced by norepinephrine and endothelin-l in the ovine uterine artery

A comparative study of adrenal medullary and cardiovascular responses to haemorrhage

From the foregoing results one can conclude that the superfused rat stomach fundic strip provides a sensitive method for continuously recording the release of cate¬ cholamines into the circulation. Injection of the animal with oestrogen subcutaneously 18 hours before the experi¬ ment gave a reliable and sensitive preparation for the assay of catecholamines released from the adrenal medulla during haemorrhage. Rat ascending colon soaked in a propranolol, (1G~^i) for half an hour before use provided a sensitive assay preparation for angiotensin generated during haemorrhage.The rabbit catecholamine released from the adrenal medulla increased at the end of the period of haetaorrhage which took 5 minutes to complete, at which time the blood pressure was lowered to about 50 - 60 cuaHg. The release of catecholamine is not continuous and decreased after the haemorrhage stopped. In some experiments catecholamine level had returned to control levels before the retransfusion of the shed blood commenced, although some intrinsic compensation and rise of arterial blood pressure had occurred. The blood pressure recovered slightly during the 30 minutes resting after the bleed, partly due to sympathetic influence, but also due to the rapid entrance of tissue fluid into the circulation as shown by decrease in both packed cell volume and haemoglobin con¬ centration. Heart rate did not increase significantly in this species during haemorrhage and fell below control levels 30 minutes after haemorrhage. This might be related to the increase of plasma volume due to entry of extra-cell liar fluid. This small rise in heart rate at the beginning of haemorrhage is probably related to the high resting heart rates in these anaesthetized animals.The release of catecholamine during haemorrhage in the rabbit was not reduced by muscarinic blockade but was abolished by nicotinic blockade which Indicates most of the receptor population are nicotinic, a finding supported by the experiments with carbachol.No evidence of increase in angiotensin generation luring or after haemorrhage suggests that no significant of the rabbit release of renin from the kidney occurs during or 30 minutes after the haemorrhage procedures, during which arterial blood pressure was reduced to 50 - 80 amHg. Angiotensin infusions into the aorta in 200, 400 and even 800 ng/kg/min did not give any evidence of releasing catecholamines from the adrenal medulla nor did discrete injections of angiotensin. This was taken as evidence that in the rabbit angiotensin does not act as a catecholamine releaser from the adrenal medulla.The above findings suggest that angiotensin neither releases catecholamine in significant amounts during haemorrhage, nor does it play any part in catecholamine release during haemorrhage in the rabbit .In dogs, catecholamine rele s« from the adrenal medulla commences as blood pressure falls and before the end of haemorrhage In most experiments when blood pressure was 50 - 80 mralig. Low levels of blood pressure did not have to be sustained for the increase in catecholamines to occur. Release was continuous over the whole period (30 minutes) after the bleed, in spite of the compensatory increase in arterial blood pressure. It returned to normal level when retransfusion of the shed blood began.The heart rate of the dog increased during haemorrhage especially towards the end and returned to control level within 30 minutes after haemorrhage. The increase in catecholamines released daring haemorrhage was not affected by muscarinic blockade. In animals treated with hexamethonium however, catecholamine release Increased during haemorrhage but not to the same level as during a bleed prior to blockade. Moreover, the output of catecholamine was a transient one persisting for only a few minutes in most of the animals studied and returning back to control levels before retransfusion started. Ligation of the renal veins together with prior administration of hexamethonlum totally abolished the release of catecholamine after haemorrhage.Angiotensin blood level in the dog increased markedly after haemorrhage and only returned to control levels after retransfusion of the shed blood. Infusion of angiotensin in 200 ng/kg/min significantly increased the release of catecholamine from the adrenal medulla In a transient burst which persisted for only 2-3 minutes and not for the whole period of the infusion. It resembled the responses noted during haemorrhage after ganglionic blockade with hexamethonluw.The above results suggest that the adrenal medulla of the dog Is very sensitive to angiotensin and that angiotension liberation during haemorrhage precedes the release of catecholamine in the dog and could play an important role in the initial release of catecholamine into the circulation during haemorrhage in this animal.The species differences noted are discussed in the context of the cardio vascular control in hypotensive states

A histochemical and ultrastructural study of vascular innervation in selected human and animal tissues

A review of the factors regulating peripheral blood flow is presented and the importance of the autonomic nervous system discussed. The purpose of this project was to extend the animal studies on vascular innervation to human tissues and in this way attempt to determine some of the constraints operating in the control of blood flow in particular vascular beds. A number of techniques designed to identify autonomic subpopulations were employed. Adrenergic nerves were demonstrated by the formaldehyde-induced fluorescence and glyoxylic acid fluorescence techniques, whilst possible cholinergic nerves were shown by acetylcholinesterase staining. The electron microsoope was also used to aid identification of autonomic subpopulations through an examination of vesicle populations in nerve endings. Extensive ultrastructural studies were undertaken on non- lactating human breast and no periarteriolar axon profiles were observed. Ultra-violet fluorescence microscopy and acetylcholinesterase staining also gave negative results indicating that blood flow through the non-lactating human breast is not controlled by autonomic nerves supplying the vessels. Studies on lactating rat mammary gland revealed a rich periarteriolar adrenergic innervation and the possibility remains that such an innervation may be present in the human gland when lactating. No acetylcholinesterase stained fibres were observed around the vessels but the arteriolar walls themselves stained strongly. Human axillary lymph nodes were examined in the electron microscope and periarteriolar adrenergic nerves demonstrated

The local control of vascular tone by perivascular nerves and the endothelium: Changes after X-irradiation and in atherosclerosis

In this thesis, the local regulation of vascular tone has been studied in normal, X-irradiated and atherosclerotic blood vessels, primarily using in vitro pharmacological techniques. In the first section, the early effects of a single dose of 4500 rad X-radiation on vasomotor control was studied in the NZW rabbit central ear artery 1, 4 and 6 weeks following exposure. Sympathetic neurotransmission was attenuated by 1 week postirradiation, with a greater reduction occuring after 4 and 6 weeks respectively. A reduction in both endothelium-dependent and independent relaxations, with no change in the contractile response to NA, indicated that the relaxant efficiency of the smooth muscle was reduced following irradiation. The neuromodulatory actions of CGRP and NPY were enhanced 6 weeks following irradiation. The second section studies the development of atherosclerosis- induced alterations in local vasomotor control during maturation in the WHHL rabbit (an animal model of familial hypercholesterolemia). Animals were examined at 4, 6 and 12 months of age, with NZW rabbits being used as controls. In the WHHL rabbit ear and mesenteric arteries, endothelium-dependent relaxant responses increased with age, unlike responses in NZW rabbit arteries. Endothelium-independent relaxations in the ear and mesenteric arteries were not altered during maturation in WHHL rabbit arteries and also when compared with NZW arteries. In contrast, the thoracic aorta from 12-month-old WHHL rabbits exhibited reduced endothelium-dependent relaxant responses to ACh, which appeared to be linked to the degree of occlusion of the vessel wall by atheromatous plaque. In the WHHL rabbit basilar artery, endothelium-dependent relaxations also increased with age. However, the basilar artery was shown to differ from the ear and mesenteric arteries in that endothelium-independent relaxant responses also increased with age. In the WHHL rabbit ear and mesenteric arteries, sympathetic neurotransmission was significantly reduced when compared with NZW rabbits. In both arteries, the reduction in sympathetic neurotransmission appeared to be due to pre- as opposed to post-junctional factors. In an addendum, relaxation by substance P in the NZW rabbit mesenteric artery was found to be mediated by neurokinin-l (NK-l) receptors located on the endothelium, with substance P and [Glp6, L- Pro9]SP6-11 being selective agonists

Dual control of local blood flow by perivascular nerves and endothelial cells: changes in development, atherosclerosis and acrylamide neuropathy

This thesis examines the influence of perivascular nerves and the vascular endothelium on the local control of vascular tone with particular emphasis on how these mechanisms are affected by age and in atherosclerosis. Using the method of in vitro pharmacology, it was shown that ATP is a cotransmitter with noradrenaline (NA) in the hepatic artery of the rabbit and that the response of the vessel to the purine is mediated by ATP acting through post-junctional P2x-purinoceptors. Acetylcholine (ACh) induced a vasodilatation that was entirely dependent on the presence of an intact endothelium whereas adenosine, ATP and 2-methylthio ATP (a selective P2y-purinoceptor agonist) were shown to elicit a relaxation that was independent of the endothelium. The presence of nerve fibres containing substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) were demonstrated in the hepatic artery of the rabbit. Furthermore, it was shown that CGRP and VIP mediated a vasodilatation in the absence of endothelium whereas SP produced a relaxation that was endothelium-dependent. Changes in the reactivity of hepatic and saphenous arteries from male and female rabbits (2-36 months) in response to directly acting vasoconstrictor agents (NA, a, methylene ATP (a, meATP), KCl); endothelium-dependent vasodilator agents (ACh, SP); endothelium-independent vasodilator agents (CGRP, VIP, ATP) and transmural nerve stimulation are described. Male and female Watanabe Heritable Hyperlipidaemic (WHHL) rabbits (4-12 months) were used as a model for human homozygous hypercholesterolemia. After an initial reduction in endothelium-dependent vasodilatation, there was an increase in relaxation at 12 months, when plaques were present. Contractions to sympathetic nerve stimulation was also reduced at 12 months. The effects of acrylamide, which is regarded as a model for autonomic neuropathy, on the response of the hepatic, saphenous and basilar arteries are also described