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R W Holz

Publications and source records attributed to R W Holz.

17 recordsLinked to original sources
PubMed1992

Expression of a cDNA for a neuronal calcium channel alpha 1 subunit enhances secretion from adrenal chromaffin cells.

A synthetic oligonucleotide was used to isolate mouse brain cDNA clones coding for a brain isoform of the alpha 1 subunit of the voltage-sensitive Ca2+ channel. Twenty-six independent cDNA clones were isolated and sequenced. All the cDNA clones reported here showed high homology to the rat brain class C cDNA sequence (Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Neuron 7, 45-57). Comparison of the individual mouse brain class C (mbC) cDNA sequences indicated the presence of four regions within the alpha 1 subunit coding sequence where alternative splicing can take place in mouse brain and raise the possibility that combinatorial arrangement of these splice variants could give rise to a heterogenous class of mbC transcripts. Northern blot analysis demonstrated that mbC mRNA sequences could be detected in highest abundance in mouse heart, at lower levels in mouse brain and spinal cord, and not at all in liver or skeletal muscle. An expression vector for one isoform of the mbC alpha 1 subunit cDNA was constructed using the human cytomegalovirus promoter to direct expression, and this expression vector was used in a novel transfection assay of primary cultures of bovine adrenal chromaffin cells. Transfection of the mbC alpha 1 subunit expression vector increased the secretion of human growth hormone derived from a cotransfected human growth hormone expression vector after stimulation with elevated K+ or the dihydropyridine agonist, Bay K8644. These experiments suggest that this isoform of the mbC alpha 1 subunit is functional in the transfected chromaffin cells and that the number of Ca2+ channels is a limiting component in the secretion from chromaffin cells in culture.

Adrenal Medulla↗
PubMed1992

Quantal secretion of catecholamines measured from individual bovine adrenal medullary cells permeabilized with digitonin.

Secretion of catecholamines from individual bovine adrenal medullary cells grown in primary culture has been investigated with a carbon-fiber microelectrode placed adjacent to the cells. Oxidation of catecholamines at the electrode surface results in changes in current, which give a real-time measure of catecholamine secretion. Chemical agents are introduced to the individual cells by pressure ejection from micropipettes. When incubated in Ca(2+)-containing buffers, secretion is not observed. However, permeabilization of the cell by exposure to 20 microM digitonin for approximately 15 s results in a Ca(2+)-dependent secretion, and the contents of individual vesicles are detected in the form of sharp spikes. The rate at which spikes occur is a function of the Ca2+ concentration in the external media and reaches a maximum at 19 microM Ca2+. The area of the spikes range from 0.1 to greater than 10 picocoulombs, but the majority are less than 2 picocoulombs, corresponding to less than 6 x 10(6) molecules detected per spike. Histograms of the spike areas are essentially independent of the Ca2+ concentration, indicating that the population of vesicles which undergo exocytosis is the same for all concentrations. Exocytotic secretion can be distinguished from nonexocytotic release by analysis of the shape of the spikes.

Adrenal Medulla↗
PubMed1992

Synthetic peptides of the effector-binding domain of rab enhance secretion from digitonin-permeabilized chromaffin cells.

There is evidence that the rab class of low molecular weight GTP-binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP-binding proteins on catecholamine secretion from digitonin-permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector-binding domains of the rab, ras and ral classes of low molecular weight GTP-binding proteins and correspond to ras(33-48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca(2+)-dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19-31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced GppNHp-induced secretion 30-70%. The peptides had little or no effect in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.

Adenosine Triphosphate↗
PubMed1992

Kinetic analysis of secretion from permeabilized adrenal chromaffin cells reveals distinct components.

We have determined that there are components to the time course of Ca(2+)-dependent secretion from digitonin-permeabilized bovine adrenal chromaffin cells that can be distinguished by Ca2+ sensitivity and ATP dependence. The effects of various Ca2+ concentrations are different on the initial rates and later rates of secretion. The earliest rates (5 s) are half-maximal between 30-100 microM Ca2+ and maximal by 300 microM Ca2+. Later rates of secretion are maximal by 10 microM and decline above 30 microM Ca2+. At low Ca2+ concentrations secretion begins after a lag of several seconds. The early rates of secretion (within 1 min) are dependent on the prior effects of MgATP. MgATP primes the cells to secrete. Later rates require the continuous presence of MgATP for optimal secretion. Incubation with low concentrations of Ca2+ increases the ability of MgATP to stimulate subsequent Ca(2+)-dependent secretion. Preincubation with Ca2+ has no effect on the rapid loss of ATP-independent secretion with time after permeabilization. The data indicate that: 1) as secretion progresses in digitonin-permeabilized cells, different events become rate-limiting; 2) maximal secretion at the early times requires at least 10-fold higher Ca2+ concentrations than at later times; 3) the rate at which Ca2+ initiates secretion is concentration-dependent; and 4) Ca2+ not only triggers the final events in secretion but enhances the ability of ATP to prime secretion.

Adenosine Triphosphate↗
PubMed1992

A temperature-sensitive step in exocytosis.

We have examined the temperature sensitivity of exocytosis in digitonin-permeabilized chromaffin cells. The time course of secretion is markedly slowed by incubating the cells at 18 degrees C rather than 27 degrees C. We have previously shown that secretion has both ATP-dependent and ATP-independent components (Holz, R. W., Bittner, M. A., Peppers, S. C., Senter, R. A., and Eberhard, D. A. (1989) J. Biol. Chem. 264, 5412-5419). Reducing the temperature has no effect on ATP-independent secretion. However, cold (18 degrees C) greatly slows the ability of ATP to stimulate secretion. The ATP-requiring priming step itself is not affected by reducing the temperature since an effect of ATP can be seen after permeabilization at 18 degrees C if the cells are subsequently stimulated to secrete at 27 degrees C. When cells are permeabilized at 27 degrees C with ATP and then stimulated by Ca2+ in the absence of ATP, the secretion which was primed by ATP during the permeabilization step is inhibited 75% at 18 degrees C. Similar results are seen when ATP-dependent priming is enhanced by low concentrations of Ca2+. Thus, the temperature-sensitive step occurs after ATP and Ca2+ act to prime the cell. The temperature-sensitive step is likely to be overall rate-limiting step during the later phase of secretion, when the ATP-dependent priming process is limiting.

Adenosine Triphosphate↗
PubMed1992

Bovine chromaffin granule membranes undergo Ca(2+)-regulated exocytosis in frog oocytes.

We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine chromaffin granules, the secretory vesicles from adrenal chromaffin cells. Highly purified chromaffin granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the chromaffin granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the chromaffin granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured chromaffin cells enriched with [3H]norepinephrine-containing chromaffin granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact chromaffin granules and chromaffin granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.

Animals↗
PubMed1992

Barium and calcium stimulate secretion from digitonin-permeabilized bovine adrenal chromaffin cells by similar pathways.

We compared the characteristics of secretion stimulated by EGTA-buffered Ba(2+)- and Ca(2+)-containing solutions in digitonin-permeabilized bovine adrenal chromaffin cells. Half-maximal secretion occurred at approximately 100 microM Ba2+ or 1 microM Ca2+. Ba(2+)-stimulated release was not due to release of sequestered intracellular Ca2+ because at a constant free Ba2+ concentration, increasing unbound EGTA did not diminish the extent of release due to Ba2+. The maximal extents of Ba(2+)- and Ca(2+)-dependent secretion in the absence of MgATP were identical. MgATP enhanced Ba(2+)-induced secretion to a lesser extent than Ca(2+)-induced secretion. Half-maximal concentrations of Ba2+ and Ca2+, when added together to cells, yielded approximately additive amounts of secretion. Maximal concentrations of Ba2+ and Ca2+ when added together to cells for 2 or 15 min were not additive. Tetanus toxin inhibited Ba(2+)- and Ca(2+)-dependent secretion to a similar extent. Ba2+, unlike Ca2+, did not activate polyphosphoinositide-specific phospholipase C. These data indicate that (1) Ba2+ directly stimulates exocytosis, (2) Ba(2+)-induced secretion is stimulated to a lesser extent than Ca(2+)-dependent secretion by MgATP, (3) Ba2+ and Ca2+ use similar pathways to trigger exocytosis, and (4) exocytosis from permeabilized cells does not require activation of polyphosphoinositide-specific phospholipase C.

Adenosine Triphosphate↗
PubMed1991

Regulation of the formation of inositol phosphates by calcium, guanine nucleotides and ATP in digitonin-permeabilized bovine adrenal chromaffin cells.

Both micromolar Ca2+ and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated the formation of inositol phosphates (InsPs) in digitonin-permeabilized chromaffin cells prelabelled with [3H]inositol. The production of InsPs was potentiated by ATP. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) caused a GTP-reversible shift to higher concentrations in the Ca(2+)-concentration-response curve for the release of InsPs without changing the maximal response. GTP[S] caused a shift to lower concentrations of Ca2+ and also increased the maximal response. The effects of GTP[S] and Ca2+ were synergistic. Although as much as 80% of the InsPs were derived from phosphatidylinositol 4-phosphate (PtdInsP) or 4,5-bisphosphate (PtdInsP2), the amount of InsPs produced could be several times the total amount of PtdInsP and PtdInsP2 in the cells and was largely accounted for by a decrease in PtdIns. The levels of labelled PtdInsP and PtdInsP2 increased on stimulation with Ca2+, but decreased on stimulation with GTP[S] or the combination of Ca2+ and GTP[S]. Preincubation with Ca2+ and ATP amplified the subsequent GTP[S]-induced production of InsPs. ATP and its gamma-thio and beta gamma-imido analogues stimulated the formation of InsPs in intact cells. However, only ATP potentiated the responses to Ca2+ and GTP[S] in permeable cells. Our main conclusions are: (1) a GTP-binding protein participates in the Ca(2+)-induced production of InsPs by phospholipase C, and (2) ATP markedly potentiates the stimulated formation of InsPs, an effect with arises from its role in polyphosphoinositide synthesis and does not involve purinergic receptor activation in permeabilized cells. The data also suggest that the different effects of Ca2+ and GTP[S] on polyphosphoinositide synthesis probably contribute to the synergistic action of Ca2+ and GTP[S] on the generation of InsPs.

Adenosine Triphosphate↗
PubMed1991

Calcium promotes the accumulation of polyphosphoinositides in intact and permeabilized bovine adrenal chromaffin cells.

1. Because cellular pools of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate turn over rapidly during phospholipase C stimulation, the continuing production of inositol phosphates requires continuing synthesis from phosphatidylinositol of the polyphosphoinositides. In the present study in adrenal chromaffin cells, we examined the effects of nicotinic stimulation and depolarization in intact cells and micromolar Ca2+ in permeabilized cells on the levels of labeled polyphosphoinositides. We compared the effects to muscarinic stimulation in intact cells and GTP gamma S in permeabilized cells. 2. Nicotinic stimulation, elevated K+, and muscarinic stimulation cause similar production of inositol phosphates (D. A. Eberhard and R. W. Holz, J. Neurochem. 49:1634-1643, 1987). Nicotinic stimulation and elevated K+ but not muscarinic stimulation increased the levels of [3H]inositol-labeled phosphatidylinositol phosphate by 30-60% and [3H]phosphatidylinositol bisphosphate by 25-30%. The increase required Ca2+ in the medium, was maximal by 1-2 min, and was not preceded by an initial decrease in phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. 3. In digitonin-permeabilized cells, Ca2+ caused as much as a twofold increase in [3H]phosphatidylinositol phosphate and [3H]phosphatidylinositol bisphosphate. Similarly, Ca2+ enhanced the production of [32P]phosphatidylinositol phosphate and [32P]phosphatidylinositol bisphosphate in the presence of [gamma-32P]ATP. In contrast, GTP gamma S in permeabilized cells decreased polyphosphoinositides in the presence or absence of Ca2+. 4. The ability of Ca2+ to increase the levels of the polyphosphoinositides decayed with time after permeabilization. The effect of Ca2+ was increased when phosphoesterase and phospholipase C activities were inhibited by neomycin. 5. These observations suggest that Ca2+ specifically enhances polyphosphoinositide synthesis at the same time that it activates phospholipase C.

Adrenal Medulla↗
PubMed1978

Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential.

The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.

Adenosine Triphosphatases↗
PubMed1977

Ionic determinants of excitability in cultured mouse dorsal root ganglion and spinal cord cells.

The ionic components of the action potentials of mouse spinal cord (SC) cells and dorsal root ganglion (DRG) cells were studied in dissociated cell cultures. It was found that the action potentials of SC cells required Na+ in the medium and were blocked by tetrodotoxin (TTX) (1 micron). Action potentials of DRG cells, on the other hand, were not blocked by TTX (up to 10 micron) and were observed in Na-free media in the presence of 8 mM Ca2+. In low Na (31 mM), low Ca2+ (0.1 mM) medium, action potentials were not observed but could be obtained if the Ca2+ concentration was increased. Action potentials of DRG cells investigated in low Na concentration in the presence of 1 mM or 8 mM Ca2+ became larger in amplitude and shorter in duration when the sodium concentration was increased. Na+ has this effect even in the presence of TTX. It is concluded that the action potentials of SC cells result mainly from a TTX-sensitive Na component. The action potentials of DRG cells on the other hand have both a TTX-insensitive Na component and a Ca2+ component.

Action Potentials↗
PubMed1977

Exchange diffusion of dopamine induced in planar lipid bilayer membranes by the ionophore X537A.

The ionophore X537A causes a large increase in the [(14)C]dopamine (a catecholamine) permeability of planar bilayer membranes. Dopamine transport increases linearly with the ionophore concentration. At relatively high concentrations in the presence of dopamine, the ionophore omdices a conductance which is nearly ideally selective for the dopamine cation. However, the total dopamine flux as determined in tracer experiments is not affected by an electric field and is over 10(5) times larger than predicted from the estimated dopamine conductance. Increasing the dopamine concentration on the side containing radioactive dopamine (the cis side) saturates the dopamine transport. This saturation is relieved by trans addition of nonradioactive dopamine, tyramine, H(+), or K(+). With unequal concentrations of dopamine cis and trans (49 and 12.5 mM), the unidirectional dopamine fluxes are equal. Increasing H(+) cis and trans decreases dopamine transport. It is concluded that at physiological pH, the X537A-induced transport of dopamine occurs via an electrically silent exchange diffusion of dopamine cation with another cation (e.g., dopamine(+), H(+), or K(+)). X537A induces a Ca(++)-independent release of catecholamines from sympathetic nerves by interfering with intracellular storage within storage vesicles (R.W. Holz. 1975. Biochim. Biophys. Acta. 375:138-152). It is suggested that X537A causes an exchange of intravesicular catecholamine with a cytoplasmic cation (perhaps K(+) or H(+)) across the storage vesicle membrane.

Anti-Bacterial Agents↗
PubMed1976

Ionophores X537A and A23187. Effects on the permeability of lipid bimolecular membranes to dopamine and calcium.

X537A carries dopamine across lipid bimolecular membranes. The rate of transport increases linearly with the X537A concentration and is independent of an electric field across the membrane. The evidence suggests that the permeating species is a neutral 1:1 complex between dopamine and X537A. A23187 does not transport dopamine. The permeability of the membrane to calcium increases as the square of the X537A concentration; the transport of calcium is also increased by A23187. With both ionophores, calcium is probably transported as an uncharged complex. Neither desmethylimipramine nor cocaine alters the transport of dopamine with X537A.

Anti-Bacterial Agents↗
PubMed1975

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187.

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 muM, release both endogenous and [3-H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [3-H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca-2+-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [3-H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca-2+ into the synaptosomes and thereby initiates exocytotoc release.

Animals↗