Accumulation Of Silver Grains Research Articles

Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

Rationale: Islet-1 positive (Islet-1+) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1+ cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine (3H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of 3H-thymidine for 5days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1+ cells. Whereas Islet− non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1+ cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

New route for Tizanidine administration: a pharmacokinetics and light microscope autoradiography study

Spasticity could represent a complication of several central nervous system (CNS) disorders. Multiple sclerosis and painful paroxysmal syndromes (e.g. trigeminal neuralgia) are pathologies in which anti-spastic drugs are used to a greater extent . Baclofen, tizanidine (TIZ), benzodiazepines, dantrolene, and, more recently, gabapentin are the pharmacological agents more widely used. Baclofen, TIZ, benzodiazepines, gabapentin, clonidine but not dantrolene are active on CNS. The alpha2 adrenoceptor agonist TIZ is one of the most effective and largely used anti-spastic drugs. Oral treatment is the only route of anti-spastics administration, although it may cause problems of bioavailability and/or compliance in spastic patients with impaired deglutition. This study was designed to assess the possibility of develop a new route of administration of TIZ and to identify its targets in the spinal cord. New Zealand rabbits were treated with oral (OR , n=6), intramuscular (IM, n=6) or intranasal (IN, n=6) TIZ (3.2 mg/kg/day). Plasma concentration was measured by HPLC on samples collected at 0, 30, 60, 90, 120 and 480 min after treatment. Curves of average concentrations of TIZ vs. time were constructed. In plasma, TIZ reached a peak between 45-65 min after administration. C max was in the range of 268.33–1213.64 ng/ml for IM treatment, of 73.95–135.92 ng/ml for IN treatment and of 16.86–857.25 ng/ml for OR treatment. After pharmacokinetic studies, different spinal cord tracts were removed and used for radioligand binding assay and autoradiography. Using [3H]-RX821002 ([3H]-RX) (0.1-14nM), the Kd and Bmax for every spinal cord segment were calculated. The non-specific binding was obtained with 100µM of (-)-epinephrine. [3H]-RX sites were accumulated in the superficial laminae of dorsal horn. Dense [3H]-RX binding in control was seen over the superficial dorsal horn (laminae I-II) and centrally located lamina X. The ventral horn showed moderate levels of binding. [3H]-RX was displaced after TIZ treatment using different administration routes. The heaviest accumulation of silver grains (lowest displacement of TIZ) occurred after OR administration of the compound, the lowest after IM administration. These results suggest that both IM and IN administration of TIZ may represent routes of administration of the drug alternative to the OR one. Considering predictable adverse effects of IM treatment, the IN administration could represent the elective route to administrate this kind of drugs.

Tanycyte Pyroglutamyl Peptidase II Contributes to Regulation of the Hypothalamic-Pituitary-Thyroid Axis through Glial-Axonal Associations in the Median Eminence

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.

Nuclear and Extranuclear Estrogen Binding Sites in the Rat Forebrain and Autonomic Medullary Areas

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

Dopamine D2-like receptors in the kidney of spontaneously hypertensive rats: a radioligand binding assay and light microscope autoradiography study.

1. Dopamine D2-like receptors were investigated in sections of kidney from male spontaneously hypertensive rats (SHRs) at 6 and 14 weeks of age using radioligand binding assay and autoradiographic techniques with [3H]-spiperone as a ligand. 2. Systolic blood pressure values were slightly higher in 6-week-old SHRs in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats and considerably higher in 14-week-old SHRs in comparison with the other groups investigated. Renal dopamine levels were higher in SHRs aged 6 and 14 weeks in comparison with age-matched WKY rats. Noradrenaline concentrations were similar in 6-week-old SHRs and normotensive WKY rats, and increased slightly in SHRs aged 14 weeks. 3. The density of [3H]-spiperone binding sites was similar in SHRs and WKY rats at 6 weeks of age, and decreased in SHRs at 14 weeks in comparison with age-matched normotensive animals. Light microscope autoradiography revealed the accumulation of silver grains in the tunica adventitia, in the adventitia-media border of intrarenal arteries and within cortical tubules. A few specific silver grains were also developed in the glomerular tuft. No changes in the density and pattern of silver grains were noticeable between SHRs and WKY rats at 6 weeks of age, whereas a reduction in silver grains largely affecting vascular binding sites was observed at 14 weeks of age. 4. Renal denervation considerably decreased the density of [3H]-spiperone binding sites in sections of rat kidney, with an almost complete loss of vascular binding sites. 5. The above findings indicate the occurrence of a decrease of dopamine D2-like receptors in the kidney of SHRs with the progress of hypertension. Dopamine D2-like receptors which are mainly prejunctional are involved in the modulation of sympathetic neurotransmission in the kidney. The loss of these receptors in SHRs may contribute to the pathophysiology of hypertension.

Pharmacological characterization and autoradiographic localization of a putative dopamine D4 receptor in the heart.

1. The pharmacological profile and the anatomical localization of a putative dopamine D4 receptor were assessed in sections of rat and human atria and ventricles using combined radioligand binding and autoradiographic techniques with [3H]-spiperone as a ligand. 2. [3H]-Spiperone was bound specifically to sections of human and rat atria and ventricles. The binding was time-, temperature- and concentration-dependent, belonging to a single class of high-affinity sites. In atria, the dissociation constant value (Kd) was 0.45 nM in rats and 0.32 nM in humans, and the maximum density of binding sites (Bmax) was 31.6+/-2.9 fmol mg(-1) tissue in rats and 18.8+/-0.7 fmol mg(-1) tissue in humans. In ventricles, Kd was 0.38 nM in rats and 0.39 nM in humans, and the Bmax was 43.5+/-3.0 fmol mg(-1) tissue in rats and 56.4+/-3.2 fmol mg(-1) tissue in humans. 3. The pharmacological profile of [3H]-spiperone binding to sections of both rat and human atria and ventricles was consistent with the labelling of dopamine D2-like receptors. [3H]-Spiperone binding was more sensitive to displacement by the neuroleptic clozapine in sections of atria than of ventricles, suggesting the expression of a dopamine D4 receptor in atrial tissue. Moreover, preincubation of some sections with a dopamine D4 receptor antibody and subsequent exposure to [3H]-spiperone caused a remarkable decrease of radioligand binding to sections of atria, but only a slight reduction of binding to sections of ventricles. 4. Light microscope autoradiography revealed the accumulation of silver grains over atrial tissue within atrial myocardiocytes. A higher density of silver grains was developed in rat than in human atria. In ventricles, silver grains were accumulated primarily in cluster areas both in rats and in humans. 5. The above findings suggest the expression of a dopamine D4 receptor in rat atria, but not in ventricles. A similar site was identified in human atria. The possible relevance of a dopamine D4 receptor in the heart is discussed.

Dopamine D 2-like receptors in the rat kidney: Effect of denervation

The effects of monolateral denervation induced by renal artery occlusion on dopamine D 2-like receptors were assessed in rat kidney using radioligand binding assay and autoradiographic techniques. [ 3 H ]spiperone was used as a ligand. [ 3 H ]spiperone was bound specifically to sections of control innervated kidneys with a dissociation constant ( K d) value of 0.07±0.003 nM and a maximum density of binding sites ( B max) value of 35.4±0.16 fmol/mg tissue. Light microscope autoradiography showed the accumulation of silver grains both in the arterial tree and in cortical tubules. At the vascular level, [ 3 H ]spiperone binding sites were accumulated primarily in the adventitia and in adventitia-media transitional zone. In cortical tubules, the higher density of [ 3 H ]spiperone binding sites was noticeable in proximal convoluted tubules. A few binding sites were also found in the glomerular tuft. In denervated kidneys, noradrenaline and dopamine levels were reduced by about 89% and 60% respectively in comparison with control innervated kidneys. Denervation reduced the density of [ 3 H ]spiperone binding sites by more than 85%. In denervated kidneys, light microscope autoradiography showed the disappearance of specific vascular binding sites and a remarkable reduction of tubular [ 3 H ]spiperone binding sites. The above results indicate that the largest majority of renal dopamine D 2-like receptors labelled by [ 3 H ]spiperone is prejunctional in location.

Localisation of dopamine D 3 receptor in the rat cerebellar cortex: a light microscope autoradiographic study

The pharmacological properties and the anatomical localisation of dopamine D 3 receptor were assessed in the rat cerebellar cortex using radioligand binding techniques associated with light microscope autoradiography and 7-[ 3H]hydroxy- N,N-di-n-propyl-2-aminotetralin (7-[ 3H]OH-DPAT) as a ligand. 7-[ 3H]OH-DPAT was specifically bound to sections of rat cerebellar cortex with a dissociation constant ( K d) of 0.5 nM and a maximum density of binding sites ( B max) of 97 ± 4 fmol/mg tissue. The rank order of potency of competitors of 7-[ 3H]OH-DPAT binding and the observation that guanosine triphosphate did not affect radioligand binding suggest the labelling of a dopamine D 3 receptor. 7-[ 3H]OH-DPAT binding sites are located mainly in the molecular layer and in lesser amounts in the Purkinje neuron layer, primarily within the cell body of Purkinje neurons. No specific accumulation of silver grains was observed in the granule neuron layer or in the white matter of the cerebellar cortex. The localisation of a putative dopamine D 3 receptor within Purkinje neurons suggests that this site may have functional relevance in the cerebellar cortex.

Autoradiographic localization of dopamine D2-like receptors in the rat adrenal gland.

The pharmacological profile and the anatomical localization of dopamine D2-like receptors were studied in sections of the rat adrenal gland using combined radioligand binding and autoradiographic techniques with [3H]-spiroperidol as a ligand. [3H]-Spiroperidol was bound to sections of the rat adrenal gland in a manner consistent with the labelling of dopamine D2-like receptor sites. The binding was time-, temperature- and concentration-dependent and of high affinity with a dissociation constant (Kd) value of 1.6 +/- 0.04 nM and a maximum density of binding sites (Bmax) of 60 +/- 3.6 fmol/mg tissue. Experiments on the pharmacological specificity of [3H]-spiroperidol binding to sections of the rat adrenal gland suggest the labelling of dopamine D3 and/or D4 receptors. The presence of dopamine D3 and D4 receptors in the rat adrenal gland was confirmed by the demonstration of a specific binding for the D3 radioligand [3H]-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (DPAT) and for the D4 radioligand [3H]-clozapine. Light microscope autoradiography showed the highest accumulation of silver grains which correspond to [3H]-spiroperidol binding sites in the rat adrenal medulla. In the adrenal cortex, where density of silver grains is about 40% lower than in the medulla, the radioligand is accumulated primarily in the zona glomerulosa and to a lesser extent in the zona reticularis. These findings suggest that dopamine D2-like receptor sites in the rat adrenal gland cortex are primarily involved in the modulation of catecholamine secretion from the medulla and of aldosterone secretion from the cortex. The possible relevance of the occurrence of dopamine D3 and D4 receptor subtypes in the adrenal gland is discussed.

Cutaneous distribution of orally administered itraconazole in guinea pigs, studied by autoradiography

AbstractBackground Itraconazole is an orally active antifungal lipophilic drug with high affinity for sebum and keratinocytes.Aim The aim of the study was to investigate the routes of percutaneous egression and the sites of cutaneous action/storage of 3H‐labelled itraconazole in an experimental animal model.Material and methods Hartley guinea pigs were dosed orally by gavage for 2 consecutive days at 10 mg/kg/day 3H‐itraconazole dissolved in polyethylene glycol 200. At various time points after the second gavage, small full‐thickness samples of dorsal skin were excised and processed using dry‐apposing autoradiography.Results Our research revealed an accumulation of silver grains over the epidermis and its appendages (hair follicle and sebaceous gland), the dermal fat tissue and the hypodermal muscle tissue on tissue sections at 6, 24 and 48 h time intervals, whereas at the remaining time points (96, 168 and 336 h) the silver grain density was markedly decreased to background level.Discussion The present histological distribution data support the current method of oral treatment with itraconazole as the treatment schedule of choice to eradicate infective organisms residing in the deeper skin layers.

Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.

3H]GABA uptake and GABA localization in mucosal endocrine cells of the rat stomach and colon.

The aim of this study was to characterize the distribution of GABAergic cells in the rat gut mucosa. Thin and/or thick serial sections of segments of rat antrum and distal colon were treated for autoradiographic localization of sites of [3H]GABA (50 nM) high-affinity uptake, or GABA immunoreactivity. Dense accumulations of silver grains were localized to a discrete population of granulated mucosal cells. These appeared to be D-type endocrine cells. These gut regions also displayed strongly GABA-immunoreactive mucosal cells. These results confirm the presence of GABAergic cells in the rat antral mucosa, and reveal that [3H]GABA is accumulated by mucosal endocrine cells in the rat colon.

Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro-TRH-derived peptide in a subpopulation of somatotrophs.

We have previously reported the presence of authentic pro-TRH-derived peptides in cultured anterior pituitary (AP) cells. The present studies were undertaken to determine whether pro-TRH mRNA could be demonstrated in the AP and to elucidate the cell type expressing pro-TRH. AP cells were cultured for up to 18 days, during which time the content of both TRH and prepro-TRH-(25-50) rose significantly (P < 0.01). In contrast, the cellular contents of LH, FSH, TSH, and ACTH fell significantly (P < 0.01), whereas that of GH increased by 45.9% (P < 0.05). Northern blot analysis revealed that the levels of pro-TRH mRNA extracted from AP cells (18 days in culture) were similar to those in hypothalamic tissue from adult male rats, indicating a high relative abundance of this mRNA in the AP. In situ hybridization experiments showed a dense accumulation of silver grains over a subpopulation of cultured AP cells. A combination of in situ hybridization for pro-TRH mRNA and immunocytochemistry for pituitary hormones revealed colocalization of pro-TRH mRNA and GH in a subpopulation of somatotrophs. No colocalization with LH-, TSH-, PRL-, or beta-endorphin-containing cells was observed. Immunocytochemistry at the electron microscopic level demonstrated that prepro-TRH-(25-50) was contained in a subpopulation of secretory granules in AP cells expressing this pro-TRH-derived sequence. These studies demonstrate that pro-TRH mRNA is present in cultured AP cells in high concentration and that the pro-TRH gene is expressed within a subpopulation of somatotrophs.

Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro- TRH-derived peptide in a subpopulation of somatotrophs

We have previously reported the presence of authentic pro-TRH-derived peptides in cultured anterior pituitary (AP) cells. The present studies were undertaken to determine whether pro-TRH mRNA could be demonstrated in the AP and to elucidate the cell type expressing pro-TRH. AP cells were cultured for up to 18 days, during which time the content of both TRH and prepro-TRH-(25-50) rose significantly (P < 0.01). In contrast, the cellular contents of LH, FSH, TSH, and ACTH fell significantly (P < 0.01), whereas that of GH increased by 45.9% (P < 0.05). Northern blot analysis revealed that the levels of pro-TRH mRNA extracted from AP cells (18 days in culture) were similar to those in hypothalamic tissue from adult male rats, indicating a high relative abundance of this mRNA in the AP. In situ hybridization experiments showed a dense accumulation of silver grains over a subpopulation of cultured AP cells. A combination of in situ hybridization for pro-TRH mRNA and immunocytochemistry for pituitary hormones revealed colocalization of pro-TRH mRNA and GH in a subpopulation of somatotrophs. No colocalization with LH-, TSH-, PRL-, or beta-endorphin-containing cells was observed. Immunocytochemistry at the electron microscopic level demonstrated that prepro-TRH-(25-50) was contained in a subpopulation of secretory granules in AP cells expressing this pro-TRH-derived sequence. These studies demonstrate that pro-TRH mRNA is present in cultured AP cells in high concentration and that the pro-TRH gene is expressed within a subpopulation of somatotrophs.

Cellular Quantification of Tyrosine Hydroxylase in the Rat Brain by Immunoautoradiography

We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a 35S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.

Timely and topologically defined protein synthesis in the peri-implanting mouse endometrium revealed by light and electron microscopic radioautography.

Many efforts have been made to correlate the morphological and biochemical evidences with changes on physiological state of the uterus during activation of the implantation window. However, the exact mechanism involved in such a phenomenon remains to be determined. The present experiment used the radioautographic approach to determine whether, chronological and/or topological variations of proteins synthesis occur in the peri-implanting endometrium. Pregnant mice submitted to ovariectomy, received exogenous supply of nidatory estradiol. After 0 to 18 hrs. of time-lapsed estradiol effects, each animal received intraperitoneally, 1 hr. pulse 3H-leucine. The uterine fragments embedded in epoxy resin, were processed for light and electron microscope radioautography. The pattern of 3H-leucine incorporation in the luminal epithelium varied according to their relation with the blastocyst present in the uterine lumen. The highest incorporation ratio was seen in the epithelium just around the conceptus 6 hrs. after estradiol treatment, while in the cells localized at interimplantation site no peak of incorporation was seen. At ultrastructural level, cell organelles involved in protein synthesis were found to be labelled. Accumulation of silver grains occurred at apical portion of the epithelial cells showed accumulation of silver grains after 6 hrs. of estradiol treatment, but not on cell surface. The endometrial stromal cells localized around the blastocyst also showed a peak of 3H-leucine incorporation 6 hrs. after estradiol, but not in the cells localized at interimplantation sites. No increased labeling was seen on the components of extra-cellular matrix at ultrastructural level. The present radioautographic experiment showed that epithelial and stromal cells localized in the endometrium of implantation chamber, changed their pattern of protein synthesis under nidatory estradiol effects. This evidence suggests that the phenomenon of implantation window induced by estradiol, is not a systemic response of the whole pregnant endometrium. The activation involves only a specific population of the endometrial cells localized just around the conceptus.

Dopexamine hydrochloride in the human kidney: localization, receptor binding and effect on 3'-5'-cyclic adenosine monophosphate generation.

Dopexamine hydrochloride is a synthetic dopamine analogue recently developed to improve myocardial and renal performance in patients suffering from low cardiac output states. The present study was designed to assess the pharmacological profile and the anatomical localization of [3H]-dopexamine binding sites within the human nephron. The effects of dopexamine on the 3'-5'-cyclic adenosine monophosphate (cAMP)-generating system in membrane particles of the human kidney were also investigated. It was found that [3H]-dopexamine was specifically bound by sections of the human kidney. The binding was partly inhibited by the DA-1 receptor antagonist SCH 23390, by the beta 2-adrenoceptor antagonist ICI 118,551 and by the DA-2 receptor antagonist domperidone. The 3 antagonists in combination reduced [3H]-dopexamine binding to nonspecific values. Light microscope autoradiography revealed the accumulation of silver grains, corresponding to [3H]-dopexamine binding sites, within the different portions of the human nephron. The incubation of sections of the human kidney with [3H]-dopexamine plus SCH 23390 caused a remarkable reduction in the density of silver grains within the proximal tubule and the macula densa and a moderate decrease in the density of silver grains within the ascending and descending limbs of the loop of Henle as well as within the distal tubule. The incubation of sections of human kidney with [3H]-dopexamine plus ICI 118,551 caused a pronounced reduction in the density of silver grains within the glomerulus, the limbs of the loop of Henle, the distal tubule and the medullary collecting tubules and a moderate decrease in the density of silver grains within the macula densa and the proximal collecting tubule.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct demonstration of dopamine D1-like receptor sites in the ciliary body of the rabbit eye by light microscope autoradiography.

The pharmacological characteristics and the anatomical localization of [3H]-SCH 23390 in sections of the ciliary body of the rabbit eye were analyzed using a radioreceptor assay and autoradiographic techniques. [3H]-SCH 23390 was bound to sections of rabbit ciliary body in a manner consistent with the labelling of D1-like receptor sites. The dissociation constant (Kd) was 0.62 nmol/l, while the maximum binding capacity (Bmax) was 117 +/- 9 fmol/mg tissue. Light microscope autoradiography revealed [3H]-SCH 23390 binding sites within the epithelium of the ciliary processes, which is the ocular structure involved in the secretion of aqueous humor. No specific accumulation of silver grains was noticeable within the iridocorneal angle, which is the structure involved in the outflow of aqueous humor. These findings suggest that the rise in intraocular pressure caused by D1 receptor agonists is probably mediated by an increase of aqueous humor formation rather than by an inhibition of the outflow of aqueous humor.

Autoradiographic demonstration of in vivo 125I-growth hormone-releasing hormone (GHRH) binding by human GH-secreting pituitary adenomas transplanted on athymic nude mice

This study was designed for the purpose of investigating a method for in vivo tumor labelling of human GH (hGH) secreting pituitary adenomas. Pituitary adenoma tissue removed from four acromegalic patients was transplanted into 62 athymic nude mice. After positive GHRH stimulation tests 125I-GHRH(1–44) NH2 was injected intravenously (i.v.) in ten nude mice. 10 min after 125I-GHRH injection, the nude mice were sacrificed, the transplants excised and prepared for light microscopical autoradiography. The mouse pituitary and skeletal muscle specimens served as controls. After the i.v. injection of 125I-GHRH we observed a marked accumulation of silver grains within the adenoma tissue indicating tumor labelling.This study is a first step in investigating a new method for labelling small residues of hGH secreting pituitary adenomas intraoperatively.

Vasoactive intestinal polypeptide receptors in rat cerebral vessels: an autoradiographic study.

1. Localization and pharmacological properties of the vasoactive intestinal polypeptide (VIP) receptors in rat circle of Willis arteries and in the arteries of pial-arachnoid membrane were studied using light microscope autoradiography combined with radioreceptor binding techniques. 2. [125I]-VIP was specifically bound to sections of rat cerebral arteries with a dissociation constant value of 0.5 nM and a binding site density of 80 fmol mg protein-1. Radioreceptor binding experiments revealed that the binding characteristics of [125I]-VIP were consistent with the labelling of specific VIP receptors. The rank order of potency of various substances tested to inhibit [125I]-VIP binding was the following: VIP greater than peptide histidine methionine greater than secretin greater than glucagon. 3. Light microscope autoradiography revealed the localization of [125I]-VIP binding sites in the medial layer of circle of Willis and pial arteries. Quantitative determination of [125I]-VIP binding site density in the different circle of Willis arteries demonstrated a higher accumulation of silver grains in the anterior than in the posterior cerebral arteries. Pial arteries are richer in VIP receptor sites than circle of Willis arteries. 4. These results suggest that the physiological neurogenic vasodilation elicited by VIP on cerebral arteries is mediated by the interaction with specific receptor sites located primarily within cerebral vessels structures involved in the control of cerebrovascular resistances.