Norepinephrine and dopamine: assay by mass fragmentography in the picomole range.

Gas chromatography-mass spectrometry makes possible the simultaneous measurement of norepinephrine and dopamine in concentrations of 0.1-milligram tissue samples. Specificity of the assay is confirmed both by the retention time of the compound and by the mass to charge ratio of the fragments recorded. The sensitivity is of the order of 0.5 picomole, and linearity of the response is maintained up to at least 200 picomoles.

Octopamine: normal occurrence in sympathetic nerves of rats.

Octopamine has been identified in several organs of normal rats by means of a sensitive enzymatic assay. It is localized within the sympathetic nerve endings.

Deoxyribonucleic acid of Cancer pagurus. IV. Elution behaviour on hydroxyapatite chromatographic column.

Fractionation of native DNA on hydroxyapatite columns depends, when flat and continuous gradients are used, on the base composition, GC-rich fractions being eluted in the first fractions. Crab satellite DNA behaves abnormally : the first eluted fractions are enriched in poly d(A-T).d(A-T) instead of GC as usual. It amy be suggested that these differences in the behaviour could be attributed to the fact that the secondary structure of crab DNA satellite is different from the secondary structure of the main DNA component.

Ultrastructural cytochemistry of biogenic amines in nervous tissue: methodologic improvements.

A new fixation method has been developed for localizing biogenic amines in nervous tissue. The method is a modification of the chromaffin reaction in which all fixation steps are buffered with mixtures of sodium chromate and potassium dichromate. In this way the fixation and cytochemical reaction are carried out almost simultaneously. Using the rat vas deferens as a model tissue, it was found that the preservation of electron dense (chromaffin) cores in the vesicles of adrenergic nerve terminals depended on several factors: a short primary fixation using low concentrations of aldehydes, the presence of the chromate/dichromate buffer during all fixation steps and, finally, a long incubation period in a slightly acidic (pH 6.0) solution of this buffer before postfixation in osmium tetroxide. Using this method it was possible to identify not only small and large dense-cored vesicles as storage sites for amines but also a tubular reticulum (neuronal endoplasmic reticulum), the latter especially in nerve terminals of mesenteric arteries and iris. Biogenic amines were also visualized in sympathetic ganglion cells and in the central nervous system e.g., supraependymal nerve terminals, tissues that up to now proved the most difficult in terms of amine localization. In all the tissues examined the cytochemical reaction was highly selective and present in well preserved tissue, which is a significant advance over previously available techniques. It therefore offers new opportunities for further studies on the role of biogenic amines as neurotransmitters.

Histochemical localization of glutathione in tissues.

A histochemical method has been developed for the localization of glutathione (GSH) in frozen sections from various tissues including liver, lung, kidney, testis and eye. The reliability and specificity of the method has been investigated by comparing the rates of reaction in tissue and gelatin sections and after depletion of GSH in liver by diethyl maleate. In principle, the method is based on the formation of an irreversible complex of mercury orange with the --SH group of GSH. A 5-min staining period was found to be optimal for staining the --SH group of GSH. In brief, frozen sections 8 mu thick are stained with a 50 muM solution of mercury orange dissolved in toluene, counterstained in 0.05 per cent methylene blue and mounted in Histoclad. Pretreatment of the sections with fixatives or drying them in air completely prevented the staining. In hepatic lobules the brick red granules of the GSH mercury orange complex were distributed uniformly, whereas in other tissues they were not uniform. The GSH staining was localized in the proximal convoluted tubules in the cortex of the kidney, the interalveolar epithelial cells of lungs, the epididymis and the capsule of testis, epithelial cells of vas deferens and the periphery of the lens.

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes.

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.

Distribution of soltriol [1,25(OH)2-vitamin D3] binding sites in male sex organs of the mouse: an autoradiographic study.

After injection of [3H]-1,25(OH)2-vitamin D3 (soltriol), nuclear labeling is found in Sertoli cells of testes, being highest at the stage of spermiosis, in epithelium of efferent ductules and caput epididymidis and in connective tissue cells of epididymis, in lamina propria and muscular sheath of deferent duct, and in epithelium and muscular sheath of dorsal and ventral prostate of the mouse. This labeling pattern is characteristic for [3H]-soltriol and differs from that for [3H]-dihydrotestosterone and [3H]-estradiol, although with overlap. The nuclear labeling with [3H]-soltriol suggests an action of the hormone on certain processes during spermatogenesis, on sperm maturation, on epididymal fluid resorption, and on secretion and transport of spermatozoa.

The effect of reserpine treatment and decentralization on the ion distribution in the vas deferens of the guinea-pig.

1. The ion distribution in the vas deferens of the guinea-pig was determined following reserpine treatment and decentralization, procedures known to increase the sensitivity of this muscle to drug stimulation.2. There were no significant changes in the estimated intracellular ion concentrations following the two procedures. The half-times of (42)K and (36)Cl effluxes were also unaltered.3. As far as ion content is concerned there is no basis for suggesting an altered resting membrane potential in supersensitive smooth muscle.

The physiological relevance of low agonist affinity binding at opioid mu-receptors.

1. Inhibition constant (Ki) were determined for a range of opioid standards using two binding assays; [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-GLYOL) binding to guinea-pig brain membranes in HEPES buffer and [3H]-naloxone binding to rat whole brain membranes in Krebs/HEPES buffer. 2. These values were compared with affinity measurements determined by antagonism of GLYOL on the rat isolated vas deferens preparation and by the receptor occlusion technique of Furchgott on the guinea-pig ileum longitudinal muscle, myenteric plexus preparation. 3. Agonists demonstrated markedly reduced binding affinity in the [3H]-naloxone binding assay where binding was conducted in the presence of sodium. 4. A strong correlation was obtained between Ki values from the [3H]-naloxone binding assay and affinity values determined in both isolated tissue preparations. Ki values obtained from [3H]-GLYOL binding did not correlate well with affinity data determined by isolated tissue techniques. 5. These findings suggest that functionally relevant receptors exhibit low agonist affinity.

Action of 6-hydroxydopamine on lamb sympathetic ganglia, vas deferens and adrenal medulla: a combined histochemical, ultrastructural and biochemical comparison with the effects of reserpine.

1. The effects of a single dose of 6-hydroxydopamine (6-OHDA) compared with those of chronic reserpine treatment were studied in lamb sympathetic neurones and adrenal medulla by a combination of fluorescence histochemistry, electron microscopy and radiochemical assay.2. In sympathetic ganglia, 6-OHDA produced a rise in noradrenaline concentration within 24 h, and falls in tyrosine hydroxylase and monoamine oxidase activities, whereas reserpine caused a fall in noradrenaline, a rise in tyrosine hydroxylase activity and no change in monoamine oxidase activity. The fluorescence of intra- and postganglionic axons increased greatly within 24 h of 6-OHDA, and there was a corresponding accumulation of large dense-core vesicles within many axons whose neurotubules were disrupted. The changes were almost reversed after 3 weeks.3. In the vas deferens, the concentration of noradrenaline and tyrosine hydroxylase and monoamine oxidase activities had all fallen 24 h after 6-OHDA treatment and had started to recover 3 weeks later. In the adrenal medulla, 6-OHDA did not alter NA concentrations but increased tyrosine hydroxylase activity whereas reserpine depleted noradrenaline and increased tyrosine hydroxylase activity.4. The changes produced in sympathetic ganglia by 6-OHDA may be due both to a direct action on the axoplasmic transport of noradrenaline containing vesicles and indirectly to the reaction of the neurones to loss of the integrity of their axons.

Factors affecting the extraneuronal inactivation of noradrenaline in cardiac and smooth muscle.

1. The relation between density of adrenergic innervation, noradrenaline (NA) accumulation (as seen with the fluorescence histochemical method) in tissues incubated in a high concentration of NA, and monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) activities, were examined in a wide range of tissues from different species.2. Evidence was obtained to support the proposal that accumulation of NA in the non-innervated smooth muscle of the human umbilical artery and chick amnion is associated with very low activities of COMT within muscle cells.3. The wide variation in tissue accumulation of NA in adrenergically innervated muscles was confirmed. For example, in the rabbit atrium and rat vas deferens, there was high NA accumulation in vascular smooth muscle but not in other muscle cells. In the mouse vas deferens there appeared to be preferential NA accumulation in the outer longitudinal muscle in comparison with the circular muscle. In the ventricle of the rat and mouse individual muscle cells showed different degrees of accumulation of NA. Many unidentified fluorescent cells were revealed in the submucosa of the guinea-pig ureter following loading with NA. The highest activities of COMT were found in the rat vas deferens and the lowest in the rabbit vascular tissues; the highest activity of MAO was found in the guinea-pig ileum, and the lowest in the rat aorta.4. No simple relation between tissue activities of MAO and COMT and degree of NA accumulation was found. Possible directions for further investigation of the problem are discussed.

Affinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor with [3H]phenoxybenzamine.

In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein.

Sertoli cell origin of testicular androgen-binding protein (ABP).

In this report it is suggested that the specific adrogen-binding protein (ABP), previously shown to originate in the testis of rat and other species, is produced by the Sertoli cells. This suggestion is based upon the following experimental findings: 1) ABP was found in high concentrations in testicular efferent duct fluid but only in trace amounts in inter-tubular lymph. i) ABP could be recovered from crude preparations of testis tubules, but not from Leydig cells from the same testes. 3) Testes whose germinal epithelium had been severly damaged by gamma irradiation showed no decrease in ABP content. The transport of ABP to epididymis was also preserved as judged from the levels of ABP in caput epididymis. 4) Testes that were completely devoid of germ cells following prenatal gamma irradiation showed high levels of ABP, These high levels approached zero following hypophysectomy, but could be restored by FSH administration to the hypophysectomized animals. ABP has been well characterized and now provides a valuable experimental tool as an indicator of Sertoli cell function.

Effects of norepinephrine on cyclic nucleotide levels in the ductus deferens of the rat.

The effect of 1-norepinephrine on cyclic nucleotide levels was studied in rat ductus deferens. Norepinephrine (0.01-0.3 mM) increased cyclic AMP and cyclic GMP levels in a concentration-dependent manner. Cyclic GMP was increased by about two-fold and cyclic AMP by 30%-40% by 0.1 mM norepinephrine after 3 min. Phenylephrine (0.1 mM) increased cyclic GMP levels about twofold without affecting cyclic AMP. Cyclic GMP was maximally increased by norepinephrine or phenylephrine after 3-10 min, but was not significantly changed after 20 sec, when contraction was fully developed. Atropine did not affect the norepinephrine-induced changes in cyclic nucleotide levels; phentolamine blocked the effect of norepinephrine on cyclic GMP, and propanolol blocked that on cyclic AMP. These findings indicate that in the ductus deferens alpha-adrenergic receptors are involved in the effect of catecholamines on cyclic GMP, and beta-adrenergic receptors are involved in the effect on cyclic AMP levels. The presence of Ca-2+ was required for the effect of norepinephrine on cyclic GMP, but not on cyclic AMP.

Enkephalin contents reflect noradrenergic large dense cored vesicle populations in vasa deferentia.

Opioid peptides including met- and leu-enkephalins are co-stored with catecholamines at similar concentrations in highly purified chromaffin granule fractions from bovine adrenal medulla and large dense cored vesicle (LDV) fractions from bovine splenic nerve. An initial attempt was made to test the universality of the co-storage of enkephalins in LDVs of sympathetic nerves. Based on a number of practical considerations, vasa deferentia were chosen from seven species. Leu-and met-enkephalin contents were quantitated by radio-immunoassays and norepinephrine by HPLC with electrochemical detection. Norepinephrine contents varied 11-fold and generally paralleled the density of sympathetic innervation among the species as reported in the literature. Leu-enkephalin contents varied 26-fold, generally paralleling the percentage composition of LDVs in the respective terminal varicosities among the species and animal size (axonal length). Met-enkephalin contents varied 20-fold, generally paralleling the density of sympathetic innervation, but not the percentage LDVs. Various amounts of met-enkephalin were also likely stored in cells other than sympathetic nerves, including chromaffin-like cells, the incidence of which varied according to species. Thus, the met- to leu-enkephalin ratios varied from 1.2:1 in dog; 1.7-1.9:1 in rabbit, cat and bull; 2.9:1 in man; 8.2:1 in rat; and essentially only met-enkephalin in guinea pig. The data imply differences in the processing of preproenkephalins in the same tissue of different species as well as in different cells of that tissue.

Characterisation of the delta-opioid receptor on the hamster vas deferens.

It has recently been reported that the hamster vas deferens contains only delta-opioid receptors. We have demonstrated that three delta-receptor agonists [D-Ala2, D-Leu5]enkephalin (DADLE), [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Thr2, Leu5, Thr6] enkephalin (DTLET) appear to mediate their effects via the delta-receptor since they are readily reversed by the selective delta-receptor antagonist ICI 174864. In addition, a number of classical mu and k receptor compounds were devoid of activity in this preparation. However it was observed that some compounds such as etorphine and MR 2034 reported to possess delta-receptor affinity in other assay systems were weak or inactive on the hamster vas deferens.

Distribution of substance K (neurokinin A) in the brain and peripheral tissues of rats.

The regional distribution of substance P (SP) and substance K (SK) in the central nervous system and peripheral tissues of rats was studied. High levels of SK-like immunoreactivity (SK-LI) and of SP-like immunoreactivity (SP-LI) were found in the substantia nigra, nucleus tractus solitarius and nucleus habenula. Neonatal treatment of rats with capsaicin caused a significant decrease in the SK-LI content in the submaxillary gland, but capsaicin had no effect on SK-LI content in any area of the brain. These observations suggest that SK in the brain and peripheral tissues may have significant physiological roles.

Are there subtypes (isoreceptors) of multiple opiate receptors in the mouse vas deferens?

The hypothesis of heterogeneity of opiate receptors was tested with the electrically stimulated mouse vas deferens. The experimental approach was based on the phenomenon of tolerance development of selective opiate receptors. It was found that opioids classified as mu-receptor agonists, e.g. sufentanyl and normorphine, do not necessarily exhibit cross-tolerance to each other. The same holds true for a variety of presumed kappa-receptor agonists. On the other hand, the delta-receptor agonists under investigation displayed complete cross-tolerance. These findings give rise to an interpretation which may postulate the existence of subtypes (isoreceptors) of the already known opiate receptors.

Increase in response and number of alpha-adrenoceptors of rat vas deferens on brief pretreatment with an alpha-agonist.

The contractile response of rat vas deferens to epinephrine (E) was enhanced by preincubation with E. After pretreatment with E, there was no significant change in the ED50 of E but there was a significant increase in the maximum response (39%). There was no change in the cholinergic response after the pretreatment. alpha-Adrenoceptors in control and E-treated tissues were assessed by measuring [3H]WB-4101 binding. Treatment with E resulted in a 27% increase in the number of alpha-adrenoceptors, but no change in muscarinic acetylcholine receptors measured with [3H]quinuclidinyl benzilate. These results are discussed in relation to the response of alpha-adrenoceptors to the agonist.

Autoradiographic localization of tritium-labeled dihydrotestosterone in human vas deferens.

The human vas deferens was examined autoradiographically for the presence and distribution of androgen receptors. Samples of vas deferens from the region proximal to the testis and the region at the internal inguinal ring were incubated in vitro with tritium-labeled dihydrotestosterone ([3H]-DHT). Frozen sections of tissue were mounted on autoradiographic emulsion-coated slides and exposed for up to three weeks to demonstrate cells with nuclear accumulations of radioactive hormone. Quantitation of autoradiograms was performed with a Zeiss Videoplan morphometric analysis system. Cells in all five tissue layers of the vas deferens were able to bind androgen receptors in the nucleus, as evidenced by superimposition of silver grains over the nuclei of cells in external, middle, and internal smooth muscle layers, as well as in epithelial and subepithelial stromal cells.