Sue Piper Duckles

Substance P in the cerebral vasculature: depletion by capsaicin suggests a sensory role

Abstract Measurements of substance P immunoreactivity confirms the presence of substance P-containing nerve fibers within the wall of cerebral blood vessels of guinea pigs, cats and rabbits. Low levels of somatostatin were also found in cerebral arteries of cats and rabbits. After treatment of guinea pigs with capsaicin, substance P levels in the cerebral circulation were markedly reduced, as was substance P in the mesenteric artery. Depletion of substance P by capsaicin suggests that cerebral arteries are innervated by primary afferent neurons, with a possible role in headache or reaction to trauma.

Vasoactive intestinal peptide as a neurotransmitter in the cerebral circulation

Cat cerebral arteries exhibit a non-cholinergic neurogenic vasodilation which is absent in rabbit cerebral arteries. Levels of vasoactive intestinal peptide (VIP) measured by radioimmunoassay are correlated with the presence of a non-cholinergic vasodilation: 270 +/- 66 pmol/g in the cat anterior cerebral artery compared to 10 +/- 2.3 pmol/g in rabbit cerebral arteries. In the absence of endothelial cells, cat cerebral arteries do not relax to acetylcholine, but continue to relax to nerve stimulation. Relaxation responses to VIP also persist. These findings are consistent with the possibility that VIP mediates non-cholinergic vasodilator responses in the cerebral circulation.

AF-DX 116 discriminates between muscarinic M2 receptors of the heart and vasculature

The newly developed muscarinic antagonist, AF-DX 116, has been reported to have a higher affinity in vivo for muscarinic receptors in the heart than in the vasculature. Therefore two in vitro preparations, the rabbit ear artery and spontaneously beating right atrium, were compared. AF-DX 116 had a 29 times greater affinity for muscarinic receptors in the cardiac preparation than in the ear artery, with a pA2 in the heart of 7.42 compared to a value of 5.95 in the ear artery. Thus AF-DX 116 shows promise as an approach to differentiating sub-classes of muscarinic M2 receptors.

Somatostatin analogs with affinity for opiate receptors in rat brain binding assay

The somatostatin analogs D-Phe-Cys-D-Trp-Lys-Thr-Cys-Thr and the corresponding penicillamine compounds have been prepared and tested for their ability to displace [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin from rat brain receptors. While somatostatin and the cystine containing peptide displayed little or no preference for either receptor system, the substitution of penicillamine at position two or seven resulted in analogs that displayed opposite receptor selectivity. The substitution of tyrosine for phenylalanine at position three resulted in a large increase in opiate receptor affinity which may be related to the known requirement for a phenolic hydroxyl moiety in the rigid opiate and enkephalin systems. Conformational properties of these analogs were also examined and related to their affinity for opiate and somatostatin receptors in the rat brain.

Dihydrotestosterone Stimulates Cerebrovascular Inflammation through NFκB, Modulating Contractile Function

Our previous studies show that long-term testosterone treatment augments vascular tone under physiological conditions and exacerbates endotoxin-induced inflammation in the cerebral circulation. However, testosterone can be metabolized by aromatase to estrogen, evoking a balance between androgenic and estrogenic effects. Therefore, we investigated the effect of the nonaromatizable androgen receptor agonist, dihydrotestosterone (DHT), on the inflammatory nuclear factor-κB (NFκB) pathway in cerebral blood vessels. Cerebral arteries were isolated from orchiectomized male rats treated chronically with DHT in vivo. Alternatively, pial arteries were isolated from orchiectomized males and were exposed ex vivo to DHT or vehicle in culture medium. DHT treatment, in vivo or ex vivo, increased nuclear NFκB activation in cerebral arteries and increased levels of the proinflammatory products of NFκB activation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Effects of DHT on COX-2 and iNOS were attenuated by flutamide. In isolated pressurized middle cerebral arteries from DHT-treated rats, constrictions to the selective COX-2 inhibitor NS398 or the selective iNOS inhibitor L-nil, [L-N6-(Iminoethyl)lysine], were increased, confirming a functional consequence of DHT exposure. In conclusion, activation of the NFκB-mediated COX-2/iNOS pathway by the selective androgen receptor agonist, DHT, results in a state of vascular inflammation. This effect may contribute to sex-related differences in cerebrovascular pathophysiology.

Age-related changes in adrenergic neuronal function of rabbit vascular smooth muscle

The adrenergic control of vascular smooth muscle was compared in young and adult rabbits using a variety of in vitro techniques. Norepinephrine (NE) content and accumulation of 3H-NE were not different in blood vessels from the two age groups. In contrast, stimulation-evoked release of endogenous NE was reduced by 40-60% in vessels from the aged animals. Functional studies of smooth muscle contractions were carried out using isolated ring segments of the ear artery. There were no differences in the resting force-response relationship between vessels from young and adult rabbits. Maximum contractile responses to nerve stimulation, NE or KCl were not different in vessels from the two age groups, nor was the NE ED50. However, blockade of the neuronal uptake system with desmethylimipramine produced a greater shift in the NE concentration-response curve in vessels from the young animals compared to the shift in vessels from adult animals. This observation reflects a decline in neuronal NE uptake with age. Although maximal contractile responses to transmural nerve stimulation at 16 Hz were unchanged, responses to stimulation at lower frequencies were reduced in vessels from adult rabbits, an effect which was also enhanced when an antagonist of neuronal uptake was present. Thus, there is a decline in function of adrenergic nerves in adult animals, reflected in a decrease in stimulation-evoked NE release and a decrease in norepinephrine uptake revealed by functional studies. These two effects tend to balance each other, so that there is a small decrease in contractile response to adrenergic nerve stimulation which is exacerbated when neuronal uptake mechanisms are blocked.

Angiotensin II potentiates responses of the rabbit basilar artery to adrenergic nerve stimulation

Abstract Angiotensin II has little contractile effect on the isolated rabbit basilar artery; however, it markedly potentiates contractile responses to adrenergic nerve stimulation. This is not a post-synaptic effect of angiotensin, as responses to exogenous norepinephrine are not altered. Angiotensin increases stimulation-evoked release of norepinephrine, and this effect probably accounts for the increased response to adrenergic nerve stimulation. Since sympathetic stimulation may protect the cerebral circulation from hypertensive damage, increased responsiveness to adrenergic nerve activity produced by angiotensin may have a beneficial effect.

Specificity of capsaicin treatment in the cerebral vasculature

Capsaicin has been shown to specifically deplete substance P from primary sensory afferents, including sensory nerves innervating blood vessels of the cerebral circulation as well as other vascular beds. In order to further document the specificity of this treatment, we examined the effect of capsaicin treatment on 3 other types of nerves in the guinea pig. Four tissues were examined: cerebral arteries, the mesenteric artery, the heart and iris. Norepinephrine content was not altered after capsaicin treatment, confirming that adrenergic nerves are unaffected. As indices of cholinergic nerves, activities of choline acetyltransferase and acetylcholinesterase were also unchanged after capsaicin treatment. In addition, no significant differences in levels of vasoactive intestinal peptide in cerebral arteries and the heart were found in animals treated with capsaicin. These findings underscore the specificity of capsaicin treatment for substance P containing nerves.

Autoradiographic localization of δ opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog

A pioneering approach to the design of a ligand selective for the 8 opioid receptors was implemented by Mosberg et al. (1983) who demonstrated that the cyclic conformationally restricted enkephalin analog (2-D-penicillamine,5-D-penicillamine)enkephalin (DPDPE) displayed extremely high specificity toward the 8 opioid receptors. Recently Akiyama et al. (1985) provided the first in vitro characterization of tritium labeled DPDPE binding to 8 opioid receptors in the rat brain and neuroblastoma-glioma hybrid (NG 108-15) cells. In the present study we examined the light microscopic autoradiographic distribution of opioid receptors in the rat central nervous system employing the highly selective 8 agonist [3H]DPDPE (S.A.= 40 Ci /mmol , Amersham Corp, Arlington Heights, IL.). Male Sprague-Dawley rats (150-200 g) were killed by decapitation, and the brain was removed and coated with plastic embedding medium (OCT Compound, Lab-Tek Products) onto microtome chucks and frozen by immersion into liquid nitrogen. Saggital and coronal sections (20 #m) were cut, thawed and moun ted onto chromealum/gelatin coated glass slides and air dried at room temperature. Slide-mounted sections were preincubated for 15 min at 25°C in 50 mM TrisHCI buffer (pH 7.4 at 25°C) containing 5 mM MgC12, 2 m g /ml BSA, 20 g g / m l bacitracin and 100 mM NaC1, to reduce any endogenous opioids

Innervation of the cerebral vasculature.

With the development of specific antibodies to vasoactive peptides and application of immunohistochemistry and radioimmunoassay methods, knowledge of vascular innervation has grown rapidly. In the cerebral circulation, four possible neurotransmitters are present: norepinephrine, acetylcholine, vasoactive intestinal peptide (VIP), and substance P. There is a dense adrenergic innervation of cerebral arteries, but contractile responses to nerve stimulation or circulating catecholamines are relatively small both in vitro and in vivo. Recent studies using radioligand binding techniques indicate a lack of specific3H-prazosin binding in cerebral arteries, in contrast to other vascular beds. Thus a lack of α1-adrenergic receptors in cerebral arteries may account for weak responsiveness to sympathetic stimulation. Both VIP and acetylcholine may be vasodilator neurotransmitters, but blockade of cholinergic responses does not alter neurogenic vasodilation. The lack of specific VIP antagonists hampers efferots to explore this system more fully. Substance P-containing nerves are affected by capsaicin, supporting the hypothesis that these are primary sensory afferents, perhaps mediating pain. Future work in this area may focus on defining the pathways of these nerves and exploring the role of co-transmitters and possible interactions between nerves. With this basic information, experiments can be designed to elucidate more clearly the functional roles these nerves play.