Subcellular distribution of adenylate cyclase and cyclic adenosine monophosphate phosphodiesterase in rat and guinea-pig vas deferens

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) is assumed to play a role in catecholamine synthesis and release. In order to determine if cyclic AMP metabolism at the level of the storage granule is important in this respect the subcellular distribution of adenylate cyclase and cyclic AMP phosphodiesterase was determined in vas deferens from normal rats, castrated rats and castrated guinea-pigs. The phosphodiesterase activity was mainly found in the soluble fractions, while the adenylate cyclase was associated with sedimentable material. When vas deferens homogenates from both intact and castrated rats were subjected to sucrose density gradient centrifugation the main part of the adenylate cyclase activity was found associated with membrane fragments at 0.5–0.6 m sucrose. The distribution of adenylate cyclase activity in the density gradient parallelled that of 5′-nucleotidase, but was different from that of noradrenaline. Also in the guinea-pig, adenylate cyclase tended to have a different distribution from that of noradrenaline.The adenylate cyclase activity in, all fractions was stimulated by fluoride and guanosine triphosphate. Noradrenaline, prostaglandin E₂, 2-chloroadenosine and phenylisopropyladenosine stimulated adenyl cyclase activity in nuclear and mitochondrial fractions, but only to a small extent, if at all, in the fractions collected from the density gradient.The results do not indicate that adenylate cyclase activity in vas deferens homogenates is associated with catecholamine storage vesicles. Hence, cyclic AMP metabolism at the level of the storage granule is probably not involved in transmitter turnover.     

Cholinergic agonists and dibutyryl cyclic guanosine monophosphate inhibit the norepinephrine-induced accumulation of cyclic adenosine monophosphate in the rat cerebral cortex

Addition of cholinergic agonists, namely carbamylcholine (carbachol), acetylcholine, eserine, eserine plus acetylcholine and eserine plus choline chloride, and dibutyryl cyclic guanosine moue-phosphate inhibited the norepinephrine-induced accumulation of cyclic adenosine monophosphate in incubated slices of rat cerebral cortex. Methacholine was ineffective and atropine did not overcome the action of carbachol on the stimulation of cyclic adenosine monophosphate synthesis by norepinephrine. Carbachol stimulated the production of cyclic guanosine monophosphate in the tissue slices white norepinephrine did not influence cyclic guanosine monophosphate levels to a significant degree.The data are in keeping with the ‘Yin-Yang’ hypothesis in which under particular situations the intracellular levels of cyclic guanosine monophosphate may modulate cyclic adenosine monophosphate concentrations.     

Guanylate cyclase from bovine adrenal medulla: Subcellular distribution and studies on the effect of lysolecithin on enzyme activity

The localization of guanylate cyclase in bovine adrenal medulla was explored by preparation of subcellular organelles. Forty per cent of the activity was recovered in the soluble fraction (cytosol), and 46% in the low-speed (800g) pellet as particulate. In order to determine the exact localization of the particulate enzyme, continuous sucrose density gradient centrifugation was used. The results indicate that guanylate cyclase activity sedimented together with acetylcholinesterase and adenylate cyclase, two plasma membrane markers. Enrichment of specific activity over that present in the crude chromaffin granule fraction was identical for these three enzymes (from 4.4 to 6.2-fold). Particulate guanylate cyclase appears therefore to be a constituent of plasma membranes.The non-ionic detergent Triton X-100 and the natural phospholipid, lysolecithin, stimulated particulate guanylate cyclase activity 20-fold, but that of the soluble enzyme only 2- to 3-fold. The kinetic behaviour of particulate guanylate cyclase was not altered by treatment with lysolecithin: the Hill n coefficient for guanosine 5′-triphosphate remained close to 1.35 and the S_0.5 value to 320 μM. Among lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce activation of particulate guanylate cyclase. Incubation of membranes with phospholipase A₂ led to a 5-fold stimulation of particulate guanylate cyclase activity, while the soluble enzyme activity was not affected. These results suggest a possible role for lysolecithin in the regulation of the intracellular levels of cyclic guanosine monophosphate during or following the excitation-secretion coupling process in the medullary cell.     

Distribution of guanylate cyclase in rat cerebral cortex: neuronal, glial, capillary, pia-arachnoid and synaptosomal fractions plus choroid plexus

Guanylate cyclase activity was examined in the choroid plexus and isolated cellular fractions including crude synaptosomes of the rat cerebral cortex. Under conditions of low Mn²⁺ (0.5 mM) the enzyme activity was depressed in preparations (homogenates, 100,000 g soluble and paniculate fractions) of cerebral cortex, pia-arachnoid, choroid plexus and cerebral neuronal (perikaryal or capillary fractions. With the homogenate and soluble enzyme forms, addition of Ca²⁺elicited an activation of guanylate cyclase in all preparations except glia. Moreover, the neuronal and cortical paniculate fractions were responsive to Ca²⁺. The calcium-dependent regulator protein (calmodulin) and carbamyl choline were without stimulatory actions on any guanylate cyclase preparation. The preparations most sensitive to activation by sodium azide consisted of those containing neuronal elements, i.e. the crude synaptosomal homogenate, the paniculate neuronal fraction, and the total cortex (homogenate and particulate). Using the homogenates as a basis for comparing basal enzyme activities, the highest guanylate cyclase activity was observed in the pia-arachnoid > total cortex > choroid plexus > synaptosomes, glia > neurons >capillaries.Thus guanylate cyclase is widely distributed among the different cellular types in the brain and individual cells vary in their sensitivities to Ca²⁺, azide and hydroxylamine.     

Asynchrony in the expression of guanosine 3':5'-phosphate-dependent protein kinase by clusters of Purkinje cells during the perinatal development of rat cerebellum

The early maturation of Purkinje cells was studied by immunocytochemistry in the rat cerebellum. The antiserum against guanosine 3':5'-phosphate-dependent protein kinase used in this study has been shown previously to label specifically all Purkinje cells in the adult rat. Immunoreactive Purkinje cells are first observed at embryonic day 17, 2 days after the end of proliferation of this neuronal population. At this time, most of the labeled cells are situated in the subventricular zone, although some immunoreactive Purkinje cells have already reached the cortex. Between embryonic day 17 and birth, four clusters of immunoreactive Purkinje cells appear in each hemicerebellum. Their time course and their pathways of migration to the cortex were followed. The immunoreactive clusters are tailed by a fibre-like immunostained material. The pattern of the migrating clusters at embryonic day 19 is very similar to the pattern of the corticonuclear projection observed at birth. From comparison between sections of embryos processed either for immunocytochemistry or Cresyl Violet staining, it appears that all the Purkinje cells are not immunoreactive. Positive and negative clusters of Purkinje cells are sharply delineated, their cells never mix. Immunopositive and negative clusters of Purkinje cells coexist until postnatal day 3. However, from birth onwards, negative clusters begin progressively in a caudorostral sequence to express guanosine 3':5'-phosphate-dependent protein kinase and rapidly attain the same level of immunoreactivity as previously labeled clusters. From postnatal day 5 all the Purkinje cells are immunoreactive. It is concluded that a compartmentalization of the cerebellar cortex is present very early and is evidenced by differences in the biochemical maturation of Purkinje cells. The axons of Purkinje cells reach the deep nuclei, following the same pathways as the clusters of Purkinje cells migrating to the cortex. Therefore, the mechanisms regulating the selection of the migratory routes followed by each Purkinje cell cluster are essential for the achievement of the topography of the corticonuclear projection. The level of protein kinase immunoreactivity cannot be taken as an index of the overall maturation of Purkinje cells, because it does not always coincide with the expression of other makers of biochemical and morphological differentiation of these neurons. During the early establishment of the cerebellar maps, an asynchrony in the expression of parts of the same genotype in the Purkinje cells may help in the establishment of ordered connections.     

Excitatory amino acid receptors and guanosine 3',5'-cyclic monophosphate in incubated slices of immature and adult rat cerebellum

Addition of the excitatory amino acids l-glutamate, l-aspartate and their analogues kainate and N-methyl-d-aspartate to incubated slices of adult rat cerebellum led to large increases in cyclic GMP levels. The order of apparent potencies was kainate greater than N-methyl-d-aspartate greater than glutamate and aspartate. D-alpha-aminoadipate and Mg2-+ inhibited responses to N-methyl-d-aspartate while glutamic acid diethyl ester was most effective against those to glutamate; responses to kainate were least affected by the antagonists. The exicitant amino acids also elicited large elevations of cyclic GMP levels in slices of immature (8 day) cerebellum. Kainate was less effective than in adult but induced two responses distinguishable by their different time courses, concentration dependencies and sensitivity to antagonists. N-methyl-d-aspartate, glutamate and aspartate were 5 to 10-fold more potent than in the adult. Responses to N-methyl-d-aspartate were similarly inhibited by d-alpha-aminoadipate and Mg2+ but those to glutamate were more resistant to glutamic acid diethyl ester than in the adult. It is concluded that the accumulation of cyclic GMP in response to excitant amino acids in the adult cerebellum is mediated via the operation of receptor types showing pharmacological characteristics expected of excitatory amino acid receptors. The actions of kainate in the immature cerebellum appear to be mediated by receptors different from those on which it acts primarily in the adult.     

Regulation of 3', 5'-cyclic guanosine monophosphate content in deep cerebellar nuclei.

The 3',5'-cyclic guanosine monophosphate (cGMP) content of deep cerebellar nuclei is decreased following activation of γ-aminobutyrate (GABA) receptors by muscimol and diazepam and is increased following a reduction of GABA content caused by isoniazid. Since previous work has shown that, in deep cerebellar nuclei, GABA receptor stimulation decreases the firing rate of neurones (Ito, Yoshida, Obata, Kawai &;Udo, 1970), it appears that the cGMP content of deep cerebellar nuclei may be an index of the activity of these neurones. Neither the destruction nor the specific activation of climbing fibers changes the cGMP content of deep cerebellar nuclei. Morphine and haloperidol decrease the cGMP content of deep cerebellar nuclei without changing the activity of enzymes that metabolize cGMP in cell free preparations or by activating GABA receptors or changing GABA content. Thus, it is tempting to conclude that a decrease in mossy fiber activity could mediate the decrease in the cGMP content of deep cerebellar nuclei induced by morphine and haloperidol injected parenterally.     

Intracellular metabolism of adenosine 3′,5′-cyclic monophosphate and calcium inward current in perfused neurones of helix pomatia

The effects of intracellular administration of substances known to affect the cyclic AMP metabolism on the calcium inward currents were studied on the somatic membrane of intracellularly perfused snail neurons. During intracellular perfusion of a neurone with 130 mM Tris aspartate solution the calcium currents, unlike the sodium ones, gradually decreased. The rate of decrease was greatly dependent upon temperature. Addition to the intracellular perfusate of exogenous cyclic AMP together with ATP and Mg²⁺ ions prevented the decrease and partially or completely restored the initial value of calcium currents; afterwards their decline was re-established but at a much lower rate. Separate intra-cellular administration of each of the latter agents did not produce a noticeable restoring effect. The maximum rate and degree of restoration could be achieved at 10^?4 M cyclic AMP, 2mM ATP and 3 mM Mg²⁺. Cyclic GMP at the same concentrations as cyclic AMP did not affect the calcium currents. A similar effect to that produced by exogenous cyclic AMP was caused by fluoride anions (8 mM) added to the perfusate together with 2mM ATP and 3mM Mg²⁺. Increase of intracellular concentration of fluoride anions up to complete substitution of aspartate produced only transitory restoration of the calcium current followed by its depression.Addition of CaCl₂(10^?4–10^?3 M) to the perfusate resulted in a rapid acceleration of the calcium current decline. This process could be slowed down with intracellular introduction of the cyclic AMP phosphodiesterase inhibitor theophylline (2 mM). Introduction of cyclic AMP together with ATP and Mg²⁺ ions also prevented the blocking of calcium conductance by intracellular calcium ions.The conclusion is drawn that the functioning of the main portion of calcium ionic channel population in the somatic membrane is closely linked with the intracellular level of cyclic AMP, probably through the activity of cyclic AMP-dependent protein kinases. Lowering of the cyclic AMP level due to its washing out during intracellular perfusion, acceleration of cyclic AMP hydrolysis by activation of phosphodiesterase or termination of cyclic AMP synthesis by exhaustion of the adenylate cyclase activity leads to a rapid transition of calcium channels to a non-functioning state, probably because of progressive dephosphorylation of the channel-forming membrane proteins.     

The immunofluorescent localization of regulatory and catalytic subunits of cyclic AMP-dependent protein kinase in neuronal and glial cell types of the central nervous system

The cellular localization of the regulatory (RI and RII) and catalytic components of the cyclic AMP-dependent protein kinase in selected areas of the central nervous system was determined using a selective and sensitive immunofluorescent technique. The neuronal and glial distribution was similar for the three subunits, however, each unit exhibited differences in the pattern of subcellular localization. Whereas the catalytic unit was confined to intranuclear loci, RI and RII were identified within both the cytoplasm and nucleus, with RII in addition showing distinct staining of the nuclear membrane. A granular distribution of RI in the cell soma further distinguished the cytological localization of these two regulatory components in this cellular compartment.The contrasting distribution of the subunits suggests possible functional differences between the regulatory and catalytic components of cyclic AMP-dependent protein kinase, which are discussed with regard to the role of this enzyme in mediating the physiological effects of cyclic AMP in the central nervous system.     

Enrichment of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in cholinergic axon terminals of the Torpedo electric organ

On fractionation of intact electric organ, cAMP and cGMP are enriched in the synaptosome fraction by factors of 14 and 6 respectively. For all regions of the electromotor neuron the absolute contents of nucleotides are in the order ATP greater than cAMP greater than cGMP. However, the molar ratios of cAMP/cGMP vary between tissues. Specific contents of both cyclic nucleotides are lowest in the nerve. For cAMP, specific contents in the axon terminal and in the perikaryal region are similar. As compared to this, cGMP is considerably enriched in the presynaptic nerve ending. This points to a physiological role of cGMP in presynaptic function.     

Neurochemical and pharmacological correlates of inferior olive destruction in the rat: attenuation of the events mediated by an endogenous glutamate-like substance

The effects of a lesion of the inferior olive (the source of climbing fibres) has been investigated on a number of parameters of cerebellar excitatory amino acid function. Protoveratrine elicits a large increase in guanosine cyclic 3',5'-monophosphate levels in cerebellar slices, probably through the release of excitatory amino acids such as glutamate or aspartate, since the receptors involved are susceptible to specific antagonists. While the direct stimulatory (postsynaptic) effects of glutamate and related amino acids were not altered after loss of climbing fibres induced by 3-acetylpyridine, the guanosine cyclic 3',5'-monophosphate response to protoveratrine was markedly (40%) attenuated. Concomitant with this was a similar reduction in the high-affinity uptake of D-[3H]aspartate, a proposed marker for glutamate/aspartate terminals. Experiments to investigate the calcium-dependent, potassium-stimulated release of glutamate and aspartate, failed to reveal any reduction following acetylpyridine lesions. Although this latter finding does not exclude the possibility that these amino acids could be the transmitter of the climbing fibres, our data would rather suggest the involvement of some glutamate-like, but as yet unidentified, neuroexcitatory substance.     

Cyclic 3',5'-adenosine monophosphate levels during the developmental cycle of Trypanosoma brucei brucei in the rat

Intracellular content of cyclic 3',5'-adenosine monophosphate was determined in several strains of Trypanosoma brucei brucei during their growth in rats. In non-relapsing infections with the cloned monomorphic strain 110M and with the cloned pleomorphic strain YTatl, the amount of cyclic AMP per 10(9) trypanosomes increased from 30 to over 90 pmol as the parasitemia increased from patency to over 10(9) organisms per ml. This increase was not observed during non-relapsing infections with strain 427. During infections with strain YTatl in both immunocompetent and lethally X-irradiated rats, cyclic AMP content of the parasite increased from 20--20 pmol per 10(9) cells early in logarithmic growth to 65--70 pmol per 10(9) cells at peak parasitemia, then decreased as the transition to intermediate and short stumpy forms commenced. At crisis, basal levels were reestablished when log slender forms were the lowest percentage of the total population and intermediate and short stumpy forms predominated, suggesting a correlation between morphologic type and level of cyclic AMP per cell during fluctuations in parasitemia. Increases in intracellular cyclic AMP were measured during in vitro incubation of the parasite in medium containing potential effectors of the trypanosome cyclic AMP system. Sodium fluoride, adenosine and methyl xanthines stimulated increases in cyclic AMP content while isoproterenol, prostaglandin E1, serotonin, histamine and several trypanocidal drugs were ineffective. The results are discussed in terms of the possible regulatory role of cyclic AMP in differentiation of trypanosomes.     

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.     

Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat

The distribution of neuropeptide Y immunoreactive cell bodies in relation to various types of catecholamine-containing cell bodies in the rat brain was analyzed immunohistochemically using antisera to tyrosine hydroxylase, dopamine β-hydroxylase and phenylethanolamine N-methyltransferase. Coexistence of the peptide in catecholamine cell bodies was established by using an elution-restaining procedure.Neuropeptide Y-like immunoreactivity was observed in most noradrenergic cell bodies of the Al/Cl cell groups in the ventro lateral medulla oblongata. Similarly this peptide immunoreactivity was also observed in the majority of the adrenergic cell bodies of the C2 group. In the dorsal and dorsal-lateral part of the nucleus of the solitary tract, where a group of small adrenergic cells is present, several small neuropeptide Y immunoreactive cells were also observed. The possibility of coexistence of adrenaline and neuropeptide Y in these cells remains to be established. The majority of the noradrenergic cell bodies of the A2 group, as well as the presumptive dopaminergic cells within its ventromedial part, seemed to lack neuropeptide Y-like immunoreactivity. Many noradrenergic cell bodies of the A6 group in the locus coeruleus proper were neuropeptide Y-immunoreactive, whereas the peptide could not be observed in the subcoeruleus group. Neither the A5 and A7 noradrenergic cells in the pons, nor any of the dopaminergic cell groups in the mesencephalon and forebrain (A8–A15) seemed to contain a neuropeptide Y-like peptide.The findings indicate that central catecholamine neurons can be subdivided into distinct sub-groups based upon the coexistence of a specific peptide.     

Changes in cyclic 3'5‘-adenosine monophosphate tissue concentration and net fluid transport in the cat's small intestine elicited by cholera toxin,arachidonic acid,vasoactive intestinal polypeptide and 5-hydroxytryptamine

We have analysed tissue cyclic 3'5′-adenosine monophosphate (cAMP) concentration in different fractions of the cat's small intestinal mucosa during secretion elicited in vivo by four different secretagogues: cholera toxin (administered intraluminally), vasoactive intestinal polypeptide (VIP; given i.a.), arachidonic acid (AA; administered intraluminally) and 5-hydroxytryptamine (5-HT; given i.a.). Cholera toxin was found to increase cAMP concentration in the villi but not in the crypts. The VIP, AA and 5-HT did not influence tissue cAMP concentration despite a profuse net fluid secretion. Hexamethonium inhibited secretion elicited by cholera toxin and AA but did not significantly influence tissue cAMP concentration. There is strong evidence for the view that villus and crypt regions of the small intestinal mucosa have different functions, secretion taking place in the crypts and absorption in the villi. However, the lack of cAMP increase in the crypts reported in this study suggests that cholera toxin in this model does not reach the crypts. The results are not in agreement with a role for cAMP in mediating secretion from the crypts, but are compatible with a role of cAMP in inhibiting absorption in the villi. It is suggested that the observed fluid secretion from the crypts elicited by cholera toxin, AA and 5-HT is to a major part mediated by intramural enteric reflexes.     

The generation of rat dorsal root ganglion cell lines to identify the target of KW-7158, a novel treatment for overactive bladder

KW-7158 is a drug candidate for the treatment of overactive bladder. Although pharmacological studies have suggested that it suppresses afferent nerve conduction, its molecular target is unknown. We herein report the establishment of dorsal root ganglion (DRG) cell lines useful for identification of the target of this compound. First, we confirmed that the target exists in rat primary DRG by [³H]KW-7158 binding. To establish DRG cell lines, we used DRG from transgenic rats harboring the temperature-sensitive large T-antigen. The immortalized cells were initially screened for their expression of neuronal markers, and 72 positive clones were obtained (designated as TRD cells). Next, in order to select TRD cells expressing the target of KW-7158, we measured the binding affinity and amount of the binding sites present in each clone. Most clones expressed two binding sites, one with low affinity and one with high affinity. Differential binding of KW-7158 derivatives to each site revealed that the high affinity site is pharmacologically relevant. Therefore, we successfully identified “TRD-10” which express the largest amount of the high affinity site. These cell lines will therefore be useful tools to identify the target of KW-7158.