Christer Owman

Autonomic nerves, mast cells, and amine receptors in human brain vessels. A histochemical and pharmacological study

The studies were performed on operation material from 17- to 63-year-old patients and on fetuses at 19-23 weeks gestational age. Formaldehyde histofluorescence showed the presence of numerous perivascular adrenergic nerves around pial and intracerebral vessels, the carotid system being better supplied than the vertebral system. Cholinergic nerves, visualized by the cholinesterase technique, followed the adrenergic fibers in the plexus formations of the pial arterial system. Histamine-containing mast cells, often with a perivascular distribution, were located with the o-phthaldiadehyde method. Transmural electrical stimulation of the perivascular nerves contracted isolated pieces of pial arteries in a frequency-dependent manner, and the response was inhibited by the adrenergic nerve blocking agent, guanethidine. On the basis of the relative potency of various amines and related compounds in producing a motor response of isolated pial arteries, and the mode of inhibition caused by specific antagonists, various amine receptors could be demonstrated: adrenergic alpha-receptors (mediating contraction) and beta-receptors (dilation), cholinergic muscarinic receptors (dilation) and histamine H2-receptors (mediating dilation). Thus, the amine mechanisms demonstrated in human brain vessels appear to be principally the same of those shown in more extensive studies on laboratory animals.

Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids.

Short-chain fatty acids (SCFAs) have long been known to exert cellular effects on blood leukocytes. Acetate, propionate, and butyrate represent the most capable SCFA, inducing calcium mobilization which subsequently regulates leukocyte function in the immune system. We have cloned the previously described putative orphan G-protein coupled receptor, GPR43, and have functionally identified SCFA as the activating ligands. Acetate and propionate were found to be the two most potent ligands, although butyrate, formate, and valerate (in this order of potency) also were able to induce receptor activation. Both the human and mouse receptor homologues were found to share the same pattern of ligand activation. This finding, together with a high degree of amino acid sequence similarity between the mouse and human homologues, indicates an evolutionary conserved function. Upon ligand stimulation, the receptor mobilized intracellular calcium in both a recombinant system as well as in human granulocytes. We found the human gene to be predominantly expressed in peripheral blood leukocytes and, to a lesser extent, in spleen. We suggest the designation FFA(2)R to this second receptor activated by free fatty acids. The first-described FFAR, now named FFA(1)R, is activated by medium- to long-chain free fatty acids.

Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice.

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Identification and terminal distribution of the tubero-hypophyseal monoamine fibre systems in the rat by means of stereotaxic and microspectrofluorimetric techniques.

Summary The monoamine-containing fibres to the median eminence and the neurointermediate lobe have been studied by fluorescence microscopy in combination with microknife lesions. The origin and the course of the fibres were evaluated on the basis of monoamine accumulation proximal to the lesion and monoamine disappearance distal to the lesion. The nature of the monoamines stored in the various fibre systems was characterized with microspectrofluorimetry. Five groups of monoamine-containing axons were seen to enter the median eminence: (a) A large group of dopamine axons which originated in cells of the arcuate nuclei and the ventral parts of the anterior periventricular nuclei; they entered the fibre layer of the median eminence close to the infundibular recess. (b) A large group of noradrenaline axons which originated in areas outside the mediobasal hypothalamus; they entered the anterior median eminence from the lateral side, close to the brain surface, and intermingled with the arcuato-hypophyseal dopamine fibres in the internal layer and the deeper part of the external layer of the median eminence. (c) Two minor groups of catecholamine axons: one reached the median eminence from the anterior side, with an antero-posterior direction, probably mingled with the axons of the supraoptico-hypophyseal tract; the other ran into the arcuate nucleus with a dorso-ventral course along and close to the third ventricle; the fibres could not be traced further. (d) A small group of scattered axons containing an unidentified fluorigenic substance which differed microspectrofluorimetrically from the catecholamines and 5-hydroxytryptamine. Judging from the disappearance of monoamines distal to the lesion and from the chemical and microspectrofluorimetric analyses of the catecholamines in the median eminence and the neuro-intermediate lobe, the following conclusions concerning the terminal ramifications of the catecholamine fibre systems could be drawn: The arcuato-hypophyseal dopamine neurones give rise to one part of the terminals in the zona externa of the median eminence and to most, possibly all, terminals in the zona interna. The tubero-hypophyseal noradrenaline fibres also have their terminals in the zona externa, and they contribute to the catecholamine innervation of the neuro-intermediate lobe. Many studies on the functional role of aminergic mechanisms in the hypothalamic regulation of the pituitary functions have focussed on the presence of dopamine in the median eminence. The present investigation has demonstrated two prominent neurone systems in the hypothalamo-pituitary complex: one containing dopamine, the other containing noradrenaline. Moreover, the results suggest the involvement of a third neurone system, probably containing an indole derivative.

Contractile response and amine receptor mechanisms in isolated middle cerebral artery of the cat

Abstract On the basis of previous histochemical investigations demonstrating a rich adrenergic and cholinergic nerve supply to the main pial arteries, the contractile response of the feline middle cerebral artery to various vasoactive amines was studied in vitro . A 4 mm long segment of the artery was mounted in a rigid system allowing the registration of even very small circular contractions by a force-displacement transducer. The preparation was maintained in an aerated and thermostatically controlled (37° C) Krebs-Ringer buffer solution at pH 7.35–7.45. Dose-response curves are presented for 5-hydroxytryptamine, acetylcholine, histamine, adrenaline, noradrenaline and isoprenaline; a contractile response up to approximately 350 dyn being obtained. The contraction induced by 5-hydroxytryptamine, acetylcholine and histamine was blocked by the respective specific antagonists. From experiments with adrenergic blocking agents it was concluded that the catecholamine-induced contraction of the pial artery is mediated through a combination of alpha- and beta-adrenergic receptors.

Origins and Pathways of Cerebrovascular Vasoactive Intestinal Polypeptide-Positive Nerves in Rat

In order to clarify the origins and pathways of vasoactive intestinal polypeptide (VlP)-containing nerve fibers in cerebral blood vessels of rat, denervation experiments and retrograde axonal tracing methods (true blue) were used. Numerous VIP-positive nerve cells were recognized in the sphenopalatine ganglion and in a mini-ganglion (internal carotid mini-ganglion) located on the internal carotid artery in the carotid canal, where the parasympathetic greater superficial petrosal nerve is joined by the sympathetic fibers from the internal carotid nerve, to form the Vidian nerve. VIP fiber bridges in the greater deep petrosal nerve and the internal carotid nerve reached the wall of the internal carotid artery. Two weeks after bilateral removal of the sphenopalatine ganglion or sectioning of the structures in the ethmoidal foramen, VIP fibers in the anterior part of the circle of Willis completely disappeared. Very few remained in the middle cerebral artery, the posterior cerebral artery, and rostral two-thirds of the basilar artery, whereas they remained in the caudal one-third of the basilar artery, the vertebral artery, and intracranial and carotid canal segments of the internal carotid artery. One week after application of true blue to the middle cerebral artery, dye accumulated in the ganglion cells in the sphenopalatine, otic and internal carotid mini-ganglion; some of the cells were positive for VIP. The results show that the VIP nerves in rat cerebral blood vessels originate: (a) in the sphenopalatine, and otic ganglion to innervate the circle of Willis and its branches from anterior and caudally and (b) from the internal carotid mini-ganglion to innervate the internal carotid artery at the level of the carotid canal and to some extent its intracranial extensions.

Pharmacological Characterization of Adrenergic Alpha and Beta Receptors Mediating the Vasomotor Responses of Cerebral Arteries Ir Vitro

The adrenergic receptors in the isolated feline middle cerebral artery were characterized pharmacologically using a sensitive system for recording circular contractions in vitro. Epinephrine. norepinephrine, isoproterenol. and phenylephrine contracted the vessel in the mentioned order of potency. Together with the inhibitory patterns obtained with graded doses of piperoxan (reversible competitive inhibition) and dibenamine or phenoxybenzamine (irreversible competitive inhibition), these results showed that the contraction was mediated by alpha receptors. With piperoxan and norepinephrine, the mean value for pA2 was 7.06 and for KH1.24 × 10−7M. The mean value for KA calculated for norepinephrine before and after partial irreversible blockade of the alpha receptors with phenoxybenzamine was 1.73 x 10−6M. The norepinephrine response was not directly proportional to the amount of receptors occupied; ED50 was reached when only about 11% of the receptors were occupied and the EAm response was obtained when 75% of the receptors were occupied. Dilation was achieved only after an active tonic contraction had been induced (with 5-hydroxytryptamine) in the vessels, and the order of potency was isoproterenol > norepinephrine - epinephrine > terbutaline. Inhibition with INPEA and propranolol was competitive, as confirmed by Arunlakshana-Schild plots, showing that the dilatory response was a beta-receptor effect. The values for pA2 (8.78 and 9.17) and KH (2.31 × 10−9M and 1.77 × 10−9M) for propranolol were indistinguishable in tests with terbutaline and isoproterenol, respectively. Comparison of the relative potencies of norepinephrine and epinephrine as well as isoproterenol and terbutaline suggested that the receptors were of the beta1 type.

Short adrenergic neurons and catecholamine-containing cells in vas deferens and accessory male genital glands of different mammals

Summary1.The adrenergic innervation and catecholamine-containing cells in the internal accessory male genital organs of the guinea-pig, rat, rabbit, cat, dog and macaque has been studied with a fluorescence microscopic method for the cellular localization of catecholamines and 5-hydroxytryptamine.2.The vas deferens and most of the accessory genital glands have a very dense supply of adrenergic terminals, which is resistant to hypogastric denervation as well as to lumbosacral sympathectomy. The adrenergic innervation is restricted to the smooth muscle coats (and the blood vessels) of the organs. Secretory cells do not seem to have an adrenergic innervation.A remarkable species difference was found in the dog: The circular muscle layer of the vas deferens contain only a sparse number of adrenergic nerve fibers but a rich network of non-adrenergic fibers.3.The adrenergic fibers of the internal accessory genital organs originates from adrenergic cell bodies located close to or within the walls of these organs. Some of these cells are surrounded by adrenergic terminals, probably belonging to similar short neurons.4.In the nervous structures adjacent to the internal accessory male genital glands as well as inside some of these organs, small polymorph cells with short processes containing high amounts of catecholamines are present. Many of these cells seem to contain adrenaline.5.In some of the genital glands the occurrence of “enterochromaffin” cells has been confirmed.

Lysophosphatidic Acid Binds to and Activates GPR92, a G Protein-Coupled Receptor Highly Expressed in Gastrointestinal Lymphocytes

Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (KD = 6.4 ± 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA1-3 receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.

Calcitonin gene-related peptide is present in mammalian cerebrovascular nerve fibres and dilates pial and peripheral arteries

Calcitonin gene-related peptide (CGRP) is a novel 37-amino acid peptide occurring in neurones within sensory ganglia, in brain stem, as well as in the walls of blood vessels of peripheral organs. Pial arteries of cat showed a well-developed supply of CGRP-positive nerve fibres. The peptide was found to be a potent dilator of both pial and peripheral vessels of rabbit and cat, and of pial vessels from man. The dilatory effect was independent of the vascular endothelium and was not mediated through adrenergic, cholinergic or histaminergic smooth muscle receptors. The neurogenic vasoconstriction induced by electrical field stimulation was temporarily inhibited by CGRP, as studied in central ear arteries from rabbits. The results suggest that CGRP is a transmitter or modulator playing a role in the regulation of vascular tone.