Pharmacological characterization and autoradiographic localization of dopamine receptors in the portal vein.

1. Dopamine (DA) and DA receptor agonists exert a variety of effects on the cardiovascular system through interaction with specific DA receptors, including decreases in blood pressure and heart rate. 2. The decrease in blood pressure is due primarily to arterial vasodilation. This phenomenon is due to the stimulation of both postjunctional (D1-like or DA1) and prejunctional (D2-like or DA2) receptors causing respectively relaxation of arterial smooth muscle and decrease of the sympathetic vasoconstriction tone. 3. In view of the lack of detailed information on the existence of DA receptors in venous tissue, we have analysed D1-like and D2-like receptors in the rat portal vein using radioligand binding techniques associated with light microscope autoradiography. 4. No D1-like receptors were demonstrated in sections of the rat portal vein, whereas the D2-like receptor ligand, [3H]-spiroperidol, was bound to sections of the vein in a manner consistent with the labelling of D2-like sites. Anatomically, D2-like sites were located within the tunica adventitia, including the adventitia-media border, and in the endothelium. 5. These findings suggest the existence of D2-like but not D1-like receptor sites in the rat portal vein. D2-like sites of the tunica adventitia are probably prejunctional and involved in the modulation of sympathetic outflow. The functional significance of endothelial D2-like sites, if any, should be clarified in future studies.

Immunogold ultrastructural localization of calpastatin, the calpain inhibitor, in rabbit skeletal muscle.

The ultrastructural localization of calpastatin, the endogenous inhibitor of the neutral calcium-dependent proteases (calpains), was investigated in rabbit skeletal muscle fibers using a polyclonal antibody against the 34 kDa form of the inhibitor isolated from rabbit. Quantitative studies by pre- and postembedding immunogold techniques revealed that the distribution pattern of the specific immunoreactivity included: 1) the sarcolemma with the adjacent cytoplasm (about 1 micron wide); 2) the myofibrils; 3) the mitochondria and 4) the nuclei (condensed as well as extended chromatin). Other cell substructures, such as lysosomes and the intermyofibrillar cytoplasm, were substantially devoid of immunoreactivity. Furthermore, in accordance to previous light microscope immunohistochemical experiments, an extracellular (endomysial) localization of specific immunoreactivity was confirmed. These results favour the view, which is also supported by a series of biochemical evidences, that calpastatin in rabbit skeletal muscle is present in cell structures also containing calpains and/or their putative substrates. The above multiple patterns of distribution also suggest that the muscular calpain-calpastatin system in skeletal muscle fibers may play different physiological roles in the various subcellular compartments.

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system.

The presence of 5-hydroxytryptamine (5-HT)-like immunoreactivity (IR) was studied in the rat female reproductive system using polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations.

Changes in glutathione content and localization in rat heart as a function of age.

The influence of aging on glutathione levels and distribution in the heart was studied in male Sprague-Dawley rats of 3 (young), 12 (adult) and 24 (old) months of age using biochemical and histofluorescence techniques, respectively. Biochemical assays of reduced glutathione (GSH) in the right and left ventricles and in the septum showed a significant decrease in GSH levels in adult in comparison with young animals. No further changes were noticeable between adult and old rats. GSH histofluorescence revealed a rather homogeneous distribution of the product of histochemical reaction within both right and left atria in 3-month-old rats. In 12-month-old rats a reduction of GSH histofluorescence in comparison with younger animals was noticeable. The loss is more consistent in the epicardial portion of the right atrium and in the endocardial region of the left atrium. In the atria of 24-month-old rats GSH reactivity was homogeneously distributed throughout the atrial wall and was significantly lower than in young or adult rats. In 3-month-old rats GSH histofluorescence was slightly lower in the epicardial than in the endocardial portions of both ventricles. In adult rats a significant decrease of GSH histofluorescence was noticeable in comparison with 3-month-old rats. The loss is particularly pronounced within the endocardial region of the left ventricle. In 24-month-old rats GSH histofluorescence showed no significant differences between adult rats. However, GSH was more homogeneously distributed throughout the ventricular wall than in adult animals. The significance of these data is discussed in relation to the role that GSH plays in protecting the myocytes against free radical damage.

Endothelial dopamine DA-1 receptor sites in the rabbit pulmonary artery: autoradiographic demonstration.

Combined in vivo radioreceptor binding and autoradiographic techniques were used to characterized the pharmacological profile and to study the anatomical localization of dopamine (DA) DA-1 receptor sites in sections of rabbit pulmonary artery. [3H]R-(+)-8-chloro-2,3,4,5-tetrahydro-5-phenyl-1H-3-benzazepin-7- alhemimaleate (SCH 23390), which was used as a ligand, was bound by sections of rabbit pulmonary artery in a manner consistent with the binding of DA DA-1 sites. The Kd value was 4.75 nM, whereas the Bmax value was 78.3 +/- 5.7 fmol/mg tissue. Light microscope autoradiography demonstrated specific [3H]SCH 23390 binding sites primarily in the endothelium of the rabbit pulmonary artery. Moreover, sparse receptor sites were visualized in the medial layer. Mechanical removal of endothelium caused the disappearance of [3H]SCH 23390 binding sites showing the endothelial localization, but was without effect on the receptor sites of the medial layer. The present findings suggest that differently from systemic arteries, where DA-1 receptor sites are localized in the medial layer, probably within smooth muscle, the majority of DA-1 sites in the rabbit pulmonary artery are endothelial. The possible significance of these sites visualized in the present study for the first time is discussed.

Calpain inhibitor in rabbit skeletal muscle: an immunochemical and histochemical study.

Calpastatin, the endogenous inhibitor of calcium-activated neutral proteases (calpains; EC 3.4.22.17), was studied in the rabbit vastus lateralis muscle by means of immunochemical and immunohistochemical techniques. Immunoaffinity chromatography using an antibody raised against the 34-kDa monomer of the 68-kDa dimeric inhibitor allowed us to isolate three main proteins (130-, 100- and 80-kDa). These proteins strongly inhibited calpain activity in muscle homogenate (I50 at about 50 micrograms/ml). Immunohistochemical experiments showed that calpastatin-related immunoreactivity was present in all fibre types (oxidative, glycolytic, oxidative-glycolytic) at both surface and cytoplasmic level. However, a few (20%) of the slow-twitch, oxidative fibres (5% of the total fibres), did not contain the cytoplasmic inhibitor. Specific immunoreactivity for calpastatin was also associated with the interstitial connective tissue. These results suggest that (i) calpastatin in skeletal muscle, as in other tissues, is present as a mixture of proteins of various molecular weights and (ii) the inhibitor may act not only in the cytoplasm but also at the surface or extracellular level.

Autoradiographic localization of the gamma-aminobutyric acid type A receptor agonist 3H-muscimol in the rat superior cervical ganglion.

The anatomical localization of gamma-aminobutyric acid type A (GABA-A) receptor sites in the rat superior cervical ganglion was studied using combined radioreceptor binding and autoradiographic techniques. 3H-Muscimol was used as a ligand of GABA-A receptor sites. The binding was consistent with the labelling of GABA-A sites. The dissociation constant value was 6.4 nmol/l, and the maximum density of binding sites was 146 +/- 7.8 fmol/mg tissue. Light microscope autoradiography revealed the accumulation of 3H-muscimol mainly in superior portions of the ganglion. Binding sites are located primarily in the neuropil rather than within ganglionic neurons. It is probable that the sites revealed by autoradiography are involved in the inhibition of acetylcholine release from ganglionic neurons.

Neurotransmitters, neuroreceptors and aging.

At first glance, it is satisfying to see the progress which has been made in the study of neurotransmitters. We have learned a great deal in the last number of years. First, we have been able to identify previously unknown compounds which affect the nervous system or associated peripheral organs. We now know a great deal about the metabolism of these molecules including their synthesis and catabolism. We have learned to identify and to classify their receptors. We have learned that alterations in the effects of neurotransmitters may be responsible for certain pathologies or may be a function of normal aging. Yet, we still have far to go in our research. There are neurotransmitters still to be discovered. We need to continue our efforts because there is still a large amount of confusion in the literature, for example, far too many contradictory reports concerning the effects of age confuse rather than clarify. Possibly order may return to the literature if investigators can agree on some basic tenets. For example, we need a basic definition of old. Some research groups consider 12-month-old rats as old while other groups consider them to be young individuals. We need to have standardization of methodology so that the conclusions can have validity. Once again certain investigators use whole brain homogenates while others use only discrete portions. We need to consider whether the effect we see in our experiment is primary or secondary to aging. We can be certain that due to the aging population, the importance of basic research of age-dependent changes in neurotransmitters and neuroreceptors will increase in the future.

Dopamine receptors mediating inhibition of the cyclic adenosine monophosphate generating system in the rat renal cortex.

1. In vitro addition of dopamine (DA) or of DA-1 receptor agonists increases 3'- 5'-cyclic adenosine monophosphate (cAMP) levels through the stimulation of DA-1 receptors. 2. Although receptor binding studies suggest the existence of DA-2 receptors in the renal cortex, the presence of DA receptors negatively coupled to cAMP generation (DA-2 effect) has not been so far characterized. 3. In this study we have shown that the addition of DA plus the selective DA-1 receptor antagonist SCH 23390 to membrane fractions of rat renal cortex decreased cAMP concentration below basal levels. 4. DA-2 receptor responses were also elicited with the DA-2 receptor agonists bromocriptine and quinpirole in the absence of SCH 23390. These inhibitory effects on cAMP generation were abolished by the DA-2 receptor antagonist 1-sulpiride. 5. The above findings are indicative of the existence in the rat renal cortex of DA-2 receptors showing an effect upon cAMP generation similar to that found in the brain and in several peripheral tissues.

Autoradiographic localization of vascular dopamine receptors.

By using combined in vitro radioreceptor binding and autoradiographic techniques, the pharmacological profile and the anatomical localization of dopamine-1 (DA-1) and dopamine-2 (DA-2) receptors were assessed in rat cerebral, mesenteric and renal arteries. 3H-SCH 23390 (DA-1 ligand) was bound by the medial layer of cerebral, mesenteric and renal arteries without different density in large, medium and small sized arteries. Moreover, 3H-SCH 23390 binding sites were not sensitive to chemical sympathectomy, suggesting postjunctional localization of DA-1 receptors. 3H-Spiroperidol (DA-2 ligand) was bound primarily by the adventitial, the adventitial-medial border as well as by the intimal layer of cerebral, mesenteric and renal arteries. The accumulation of adventitial and adventitial-medial 3H-spiroperidol binding sites was higher in medium and small sized arteries than in large ones and was remarkably reduced after chemical sympathectomy. These results show prejunctional localization of DA-2 receptors and further suggest that some DA-2 binding sites are located in the arterial intima.

Age-related changes of the noradrenergic innervation of the uterine vasculature: a fluorescence histochemical study.

Age-related changes in the density of the noradrenergic perivascular plexus supplying the uterine artery and its intrauterine branches were studied in rats 4, 12 and 24 months old using the glyoxylic acid fluorescence technique. Moreover, uterine noradrenaline levels were determined by means of high pressure liquid chromatography with electrochemical detection. No changes in the density of noradrenergic innervation or in uterine noradrenaline levels were noticeable in 12 month in comparison with 4 month rats. In contrast, a significant decrease in the density of the noradrenergic perivascular plexus of uterine artery and of its intrauterine branches as well as decrease in noradrenaline concentrations of uterine horns and cervix were observed in 24 month rats. The above findings indicate an age-related impairment of uterine noradrenergic innervation.

Age-related changes of dopamine sensitive cyclic AMP generation in the rat frontal cortex.

The dopamine (DA) D-1 and D-2 receptors coupled to 3',5'-cyclic adenosine monophosphate (cAMP) generation were studied in membrane particles of the frontal cortex in young (3-month-old), adult (12-month-old) and aged (24-month-old) male Sprague-Dawley rats. Activation of D-1 receptors with DA, apomorphine or fenoldopam enhanced accumulation of cAMP in the frontal cortex of young rats. The stimulatory effect elicited by DA on cAMP generation declined by about 20% in adult rats. No further decline in cAMP accumulation was noticeable in aged animals. The response to dopaminergic agonists was blocked by the D-1 receptor antagonist SCH 29390 in the three age groups examined. The presence of D-2 receptors, negatively coupled to cAMP generation, was demonstrated by incubating frontal cortex membrane particles with SCH 23390 and then with DA. This inhibitory response, was also elicited with D-2 receptor agonists quinpirole or bromocriptine in the absence of SCH 23390 in which these compounds produced a decrease in cAMP. The decrease in cAMP caused following D-2 receptor stimulation was shown to be enhanced with age. No difference was observed between the three age groups of animals in the activation of cAMP production by forskolin. The present data suggest a selective decrease in the coupling between the D-1 receptor and cAMP generation in the frontal cortex of adult and aged rats and of an age-dependent increase in the coupling between the D-2 receptor and cAMP inhibition. The functional consequences of these biochemical changes may have important implications in the aging of the rat frontal cortex.

Autoradiographic localization of vasoactive intestinal polypeptide receptors in the rat mesenteric vascular tree.

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.

3H-muscimol binding sites within the rat choroid plexus: pharmacological characterization and autoradiographic localization.

By using combined radioreceptor binding and autoradiographic techniques, we were able to localize the GABA 'A' receptor agonist 3H-muscimol in the rat choroid plexus. Within sections of rat choroid plexus, 3H-muscimol was bound specifically with a KD of 37 nM and a Bmax of 253 pmol/mg tissue. These values are consistent with the labelling of GABA 'A' receptor sites. The autoradiographic studies demonstrated that 3H-muscimol was attached to the epithelium of the choroid plexus. The blood vessels of the plexus did not exhibit specific labelling. Examination of these data suggests the existence of GABAergic mechanisms which control cerebrospinal fluid production or flow.

Effect of dopamine on the cyclic adenosine monophosphate generating system in the rabbit internal carotid and middle cerebral artery.

The effect of in vitro addition of dopamine on the 3'-5'-cyclic adenosine monophosphate (cAMP) generating system of rabbit internal carotid and middle cerebral artery was studied. Dopamine increased cAMP levels in a dose-dependent manner in both arteries. The DA1-receptor antagonist SCH 23390 decreased the dopamine-elicited cAMP increase. On the contrary, the DA2-receptor antagonist L-sulpiride caused an increase in dopamine-dependent cAMP levels. The simultaneous addition of SCH 23390 and of L-sulpiride abolished any effect of dopamine on cAMP content. The DA2-receptor agonists bromocriptine and co-dergocrine decreased cAMP levels. The above findings indicate the existence in the rabbit internal carotid and middle cerebral artery of 2 types of dopamine receptors mediating respectively, the increase (DA1-effect) and the decrease (DA2-effect) of cAMP levels.

GABAA receptor sites modulating catecholamine secretion in the rat adrenal gland: evidence from 3H-muscimol autoradiography and in vivo functional studies.

The occurrence and distribution of specific 3H-muscimol binding sites, most probably identical with A type gamma-aminobutyric acid (GABA) receptors, were studied in sections of the rat adrenal gland by light microscope autoradiography. Specific binding was found primarily in the adrenal medulla, in association with chromaffin cells. A limited number of binding sites was also observed within the adrenal cortex. In urethane-anaesthetized hexamethonium-pretreated rats, intravenous GABA produced a set of 'excitatory' cardiovascular effects (increase in heart rate, force of contraction and blood pressure) which were mimicked by intravenous muscimol but not by intravenous baclofen, and were antagonized by pretreatment with bicuculline. The cardiovascular excitatory effects of intravenous GABA were unaffected by reserpine pretreatment, markedly reduced by administration of phentolamine plus propranolol, and almost completely abolished by adrenalectomy. Our findings indicate the presence of GABA receptor sites on adrenal chromaffin cells, whose excitation can produce changes in cardiovascular function.

Effect of vasoactive intestinal polypeptide on the cyclic adenosine monophosphate generating system in rat kidney glomeruli and cortical tubules.

The effect of in vitro addition of vasoactive intestinal polypeptide (VIP) on the 3'-5'-cyclic adenosine monophosphate (cAMP) generating system in rat kidney glomeruli and cortical tubules was studied. VIP did not stimulate cAMP accumulation in glomeruli; but VIP did stimulate, specifically and in a dose-dependent manner, cAMP concentrations in tubular membranes with the addition of GTP. These results are consistent with the existence of a functionally active VIP receptor coupled to adenylate cyclase in rat kidney cortical tubules.

Enzyme histochemistry of monoamine oxidase in the heart of aged rats.

The distribution of monoamine oxidase types A and B in the heart of young (3-month-old) and aged (26-month) Wistar rats was studied by histochemical methods. In young rats, MAO activity was higher in the left ventricle than in the right. Enzyme reactivity was present within both muscle cells and the blood vessel wall. Histochemical staining was abolished by clorgyline administration. In the heart of aged rats, we observed a very remarkable increase of clorgyline sensitive MAO activity primarily at the level of myocardial cells. The right ventricle showed the highest increase of MAO reactivity.

The noradrenergic innervation of the vasa nervorum in old rats: a fluorescence histochemical study.

The effect of ageing on the density and pattern of noradrenergic nerves in the perivascular nerve plexus supplying the vasa nervorum of the rat sciatic nerve was studied using combined catecholamine histofluorescence and quantitative image analysis techniques. The density of noradrenergic fibres around arteries and arterioles of the rat sciatic nerve vasa nervorum increased in the old animals. In contrast, no changes in perivascular nerve fibres supplying the veins and venules were found in the vasa nervorum of old rats. The increase in old age of noradrenergic innervation of arteries and arterioles of the vasa nervorum may be related to the pathogenesis of some peripheral nerve diseases.

The adrenergic innervation of rat uterine tubes in old age: a fluorescence histochemical study.

The effect of ageing on the density and pattern of adrenergic innervation of rat uterine tubes was studied using combined glyoxylic acid induced fluorescence and quantitative image analysis techniques. With age a reduction in the number of nerve fibers of the circular muscle layer of the isthmic portion was observed. No significant differences in the density of innervation were found at the level of blood vessels associated with uterine tubes.