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R Penner

Publications and source records attributed to R Penner.

At least 55 records · Page 3Linked to original sources
PubMed1989

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA.

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.

Animals↗
PubMed1989

The patch-clamp technique in the study of secretion.

One of the basic cellular functions of virtually every cell type is the exocytotic release of molecules synthesized, stored and packaged into intracellular vesicles or granules. Over decades much effort has been concentrated on elucidating the chain of events leading to exocytosis. Unfortunately, the nature of the process that ultimately induces membrane fusion is not known, nor has it been established definitively whether or not the final steps in the secretory cascade are identical in different cells. Although the fusion between vesicle and plasma membrane has been neatly documented by electron micrographs, it was only recently that the technique of time-resolved membrane capacitance measurement has provided a more detailed insight into mechanistic aspects of exocytosis, both in terms of the fusion event and the steps involved in stimulus-secretion coupling.

Adrenal Glands↗
PubMed1989

Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells.

Simultaneous patch-clamp and fura-2 measurements were used to investigate the electrical properties and receptor-mediated changes of intracellular calcium in renal juxtaglomerular cells. Here we report the presence of voltage-activated inward and outward rectifying potassium currents and the inhibition of the anomalous inward rectifying potassium current by angiotensin II (ANG-II). This action of ANG-II was mimicked by guanosine 5'-[gamma-thio]triphosphate but not by cAMP, cGMP, inositol 1,4,5-trisphosphate, or phorbol ester, suggesting that ANG-II inhibits the potassium channel directly by means of a guanine nucleotide-binding regulatory protein or by means of an unusual type of second messenger. Blocking of the inward rectifier was paralleled by membrane depolarization, but we obtained no evidence for calcium entry due to voltage-gated calcium channels in juxtaglomerular cells. Instead, under voltage clamp, ANG-II and guanosine 5'-[gamma-thio]triphosphate induced release of calcium from intracellular stores followed by a sustained phase of transmembrane calcium influx and oscillations of intracellular Ca2+ concentrations. Changes in intracellular Ca2+ concentrations were found to depend on the extracellular Ca concentration--i.e., the sustained elevation was abolished in absence of extracellular Ca, and the frequency of repetitive calcium release was directly related to the extracellular concentration of calcium. Moreover, an elevation of extracellular Ca concentration by itself induced release of intracellular calcium in the absence of other stimuli. Changes in intracellular Ca2+ concentrations were accompanied by prominent calcium-activated chloride currents, and this mechanism is inferred to be responsible for the inhibitory role of calcium in renin secretion. Intracellular application of cAMP but no cGMP inhibited ANG-II and guanosine 5'-[gamma-thio]triphosphate induced calcium mobilization in juxtaglomerular cells, being consistent with the facilitatory effects of elevated cAMP levels of renin release. The frequency of ANG-II induced oscillations was also markedly attenuated at depolarized membrane potentials suggesting effective negative feedback control of ANG-II-induced depolarization on repetitive Ca2+ transients induced by the hormone.

Angiotensin II↗
PubMed1989

Second messenger-activated calcium influx in rat peritoneal mast cells.

1. To study the regulation of calcium influx in non-excitable cells, membrane currents of rat peritoneal mast cells were recorded using the whole-cell patch-clamp technique. At the same time, intracellular calcium concentration ([Ca2+]i) was monitored via the fluorescent calcium-indicator dye Fura-2, which was loaded into cells by diffusion from the patch pipette. 2. Stimulation of mast cells with secretagogues, such as compound 48/80 or substance P, caused release of Ca2+ from internal stores. In addition, external agonists also induced influx of external calcium in 26% of the cells investigated. The agonist-stimulated Ca2+ influx was increased during membrane hyperpolarization and was associated with small whole-cell currents. 3. Likewise, internal application of inositol 1,4,5-trisphosphate (Ins1,4,5P3:0.5-10 microM) elevated [Ca2+]i due both to release of Ca2+ from internal stores and to influx of external calcium. The Ins1,4,5P3-induced influx was greater at more negative membrane potentials, suggesting that Ins1,4,5P3 opened a pathway through which calcium could enter at a rate governed by its electrochemical driving force. 4. Inositol 1,3,4,5-tetrakisphosphate (Ins1,3,4,5P4) did not induce Ca2+ influx by itself nor did it facilitate or enhance Ins1,4,5P3-induced Ca2+ entry. Calcium influx was also induced by inositol 2,4,5-trisphosphate. Since this inositol phosphate is a poor substrate for Ins1,4,5P3 3-kinase it seems unlikely that Ins1,3,4,5P4 plays a role in the regulation of the Ca2(+)-influx pathway in mast cells. 5. The Ins1,4,5P3-induced Ca2+ influx was associated with whole-cell currents of 1-2 pA or less, with no channel activity detectable in whole-cell recordings. The small size of the whole-cell current suggests either that the Ins1,4,5P3-dependent influx occurs via small-conductance channels that are highly calcium specific or that the influx is not via ion channels. 6. Agonist stimulation also activated large-conductance (ca 50 pS) cation channels, through which divalent cations could permeate; thus, these channels represent a second pathway for Ca2+ influx. The slow speed of activation of the channels by agonists, their activation by internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), and the inhibition of agonist activation by internal guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) all suggest that the 50 pS channels are regulated by a second messenger and/or a GTP-binding protein. The activity of the 50 pS channel in mast cells is not sensitive to either Ins1,4,5P3 or Ins1,3,4,5P4. Activity of the channel was inhibited by elevated [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells.

1. Stimulation of mast cells by externally applied secretagogues activated a slowly developing membrane current. With high external and low internal chloride (Cl-) concentrations, the current reversed at about -40 mV, but when external Cl- was made equal to internal Cl-, the reversal potential shifted to about 0 mV, demonstrating that the current carrier was Cl-. 2. In addition to external agonists, internally applied cyclic AMP and high concentrations of intracellular calcium [Ca2+]i could also activate the Cl- current. However, elevated [Ca2+]i produced only slow and incomplete activation. This suggests that the Cl- current is not directly Ca2+ activated. Also, activation of Cl- current by external agonists and by cyclic AMP was unimpaired when [Ca2+]i was clamped to low levels with internal ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA), indicating that elevated [Ca2+]i is not necessary for activation of the Cl- current. Although activation by cyclic AMP was faster than that produced by elevated [Ca2+]i, it still required tens of seconds; thus the effect of cyclic AMP was also likely to be indirect. 3. Internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) could also activate the Cl- current, suggesting the involvement of a G protein in the control of the current. 4. The variance associated with the Cl- current was small, and noise analysis gave a lower limit of about 1-2 pS for the single-channel conductance. The Cl- current was reduced by 4,4'-diisothiocyano-2,2'-stilbenedisulphonate (DIDS), and during DIDS blockade, the variance of the current increased. This suggests that DIDS enters and blocks the open channel. 5. Activation of the Cl- current would make the membrane potential negative following stimulation of a mast cell, thus providing a driving force for entry of external calcium via the stimulation-induced influx pathways described in the preceding paper (Matthews, Neher & Penner, 1989).

Animals↗
PubMed1989

[The importance of calcium for secretion in excitable and non-excitable cells].

Secretion via exocytosis is a process common to excitable as well as non-excitable cells. The notion that this process is entirely determined by a rise in [Ca]i is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced levels of [Ca]i. It appears appropriate to distinguish between electrically excitable and electrically non-excitable cells. In the former, a rise in [Ca]i is the triggering event for secretion, whereas in the latter, second messengers seem to induce secretion while [Ca]i acts as a modulator of the rate of secretion. Conversely, second messengers may modulate Ca-induced secretion in excitable cells.

Animals↗
PubMed1988

Regulation of calcium influx by second messengers in rat mast cells.

Biphasic increases in the free intracellular calcium concentration, consisting of a large initial transient followed by a sustained elevation, are frequently observed in non-excitable cells following stimulation. In rat peritoneal mast cells a cAMP- and Ca-activated chloride current can interact with IP3-dependent calcium influx to provide the sustained elevation of intracellular Ca concentration following transient IP3-induced release of calcium from intracellular stores. This novel combination of second messenger systems provides a flexible means to modulate calcium-dependent processes such as exocytosis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Secretory responses of rat peritoneal mast cells to high intracellular calcium.

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.

Animals↗
PubMed1988

Multiple signaling pathways control stimulus-secretion coupling in rat peritoneal mast cells.

Fura-2 and membrane capacitance measurements were performed to investigate intracellular Ca2+ concentration [( Ca2+]i) and secretory responses of rat peritoneal mast cells following secretagogue stimulation. Compound 48/80 and internally applied guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) induced transient rises in [Ca2+]i and caused membrane capacitance increases as secretion occurred. The 48/80-induced Ca2+ transients and secretory responses were blocked by guanosine 5'-[beta-thio]diphosphate and neomycin, indicating that inositolphospholipid breakdown mediated by guanine nucleotide-binding regulatory protein (G protein) plays an important role in stimulus-secretion coupling. However, pertussis toxin did not block Ca2+ transients induced by 48/80 or GTP[gamma-S], whereas secretory responses were either abolished (48/80) or developed only after a considerable delay (GTP[gamma-S]). Similar effects were obtained by perfusing cells with cAMP: (i) Ca2+ transients following stimulation with 48/80 remained unaffected by cAMP, but secretory responses were abolished; (ii) GTP[gamma-S] induced normal Ca2+ transients and degranulation in the presence of cAMP. Pretreatment of mast cells with phorbol 12-myristate 13-acetate (PMA) abolished 48/80- and GTP[gamma-S]-induced Ca2+ transients (but not inositol trisphosphate-induced Ca2+ transients), whereas secretion still occurred. At the same time, the Ca2+ requirement for secretion was reduced by PMA. These results indicate that secretion in mast cells is under control of an as yet unidentified signaling pathway that involves a G protein. This pathway is distinct from inositolphospholipid turnover and may provide the triggering mechanism for secretion, whereas the inositolphospholipid pathway serves to increase [Ca2+]i and renders the secretory process more sensitive to [Ca2+]i by activating protein kinase C. Persistent activation of protein kinase C through phorbol ester imposes negative feedback control on the inositolphospholipid pathway, whereas cAMP may inhibit the unidentified signaling pathway.

Animals↗
PubMed1988

The role of calcium in stimulus-secretion coupling in excitable and non-excitable cells.

Secretion of vesicular contents by exocytosis is a common feature of excitable (neurones, chromaffin cells, beta cells) and non-excitable cells (platelets, neutrophils, mast cells). The simplistic view that the universal mechanism controlling secretion is elevation of [Ca2+]i--whatever the source of this second messenger may be--is no longer tenable in view of recent reports demonstrating secretion at basal or even reduced [Ca2+]i. It is nevertheless clear that in excitable cells an increase in [Ca2+]i is the triggering event that induces secretion. In non-excitable cells, secretion is presumably triggered by other second messengers, although [Ca2+]i appears to act as an important modulator of the rate of secretion. Conversely, these second messenger systems may serve a regulatory function in excitable cells. Given the relative importance of [Ca2+]i in the regulation of cellular functions in excitable and non-excitable cells, it is not surprising that several mechanisms are expressed in these cells to regulate intracellular calcium concentration. The major pathway for Ca2+ in excitable cells is by voltage-activated Ca2+ channels, but release of Ca2+ from intracellular stores, via second messengers, predominates in non-excitable cells, and may also be important in excitable cells. In addition, receptor-operated channels and second messenger-gated conductances may prove to be important. All of these pathways are subject to regulation by a variety of interactive second messenger systems, which provide necessary tuning for an appropriate control of intracellular calcium level.

Animals↗
PubMed1987

Differential effects of various secretagogues on quantal transmitter release from mouse motor nerve terminals treated with botulinum A and tetanus toxin.

Electrophysiological and electron microscopic techniques were used to investigate the actions of potassium depolarization, black widow venom (BWSV), Ca2+-ionophore A 23187 and hyperosmotic solution on mouse hemidiaphragms poisoned in vitro with botulinum A toxin (BoTx) and tetanus toxin (TeTx). These neurotoxins reduced the frequency of miniature endplate potentials (m.e.p.ps) from 5/s of the control to 2/min and 21/min, respectively. High potassium (25 mmol/l) increased the m.e.p.p.-frequency at BoTx- and TeTx-poisoned endplates to 30/min and 50/s, respectively. The ultrastructure of endplates showed no obvious changes. BWSV (0.04 glands/ml) was just as effective in promoting transmitter release from BoTx-treated endplates as in control preparations. Electron micrographs revealed depletion of vesicles as well as swollen and disrupted mitochondria. When preparations were pretreated with TeTx, BWSV only moderately increased transmitter release and no alterations of the ultrastructure could be observed. At TeTx- or BoTx-poisoned endplates Ca2+-ionophore A 23187 usually produced an extreme reduction of m.e.p.p.-frequency (0.005/s), sometimes preceded by a short burst-like release. The ultrastructure of these endplates was not obviously affected. Application of hyperosmotic solution to BoTx- or TeTx-poisoned preparations further reduced the already low m.e.p.p.-frequency. These results further demonstrate that TeTx and BoTx act at different sites in the transmitter releasing process.

Animals↗
PubMed1987

Distinct sites of action of clostridial neurotoxins revealed by double-poisoning of mouse motor nerve terminals.

(1) We investigated the effects of single- and double-poisoning with tetanus toxin (TeTx), botulinum neurotoxin type A (BoTx A) and botulinum neurotoxin type B (BoTx B) on spontaneous and nerve-evoked quantal transmitter release at motor endplates of the triangularis sterni preparation of the mouse. (2) Inhibitory effects of TeTx and BoTx B on spontaneous and nerve-evoked transmitter release were very similar, except that the action of BoTx B required 500-fold lower concentrations and was less dependent on temperature. BoTx A caused stronger inhibition of quantal release than TeTx or BoTx B, but was comparatively much easier counteracted by 4-aminopyridine (4-AP). (3) In contrast to BoTx A, with TeTx or BoTx B the increase of transmitter release following onset of 50 Hz nerve stimulation was delayed for a few seconds and synaptic latencies of quanta showed large variations. This release pattern was also evident in all double-poisoning experiments, regardless of intoxication sequence. (4) Inhibition of evoked release was found to be slightly stronger with TeTx than with BoTx B, so the amount of nerve-evoked quanta released after double-poisoning with any sequence of these toxins always approached that of TeTx. In no case supra-additive actions were observed. (5) A strong reduction of evoked quanta was observed when BoTx A was applied in addition to either of the two other toxins. With reversed poisoning sequences (BoTx A - TeTx or BoTx A - BoTx B) the resulting values remained at the extremely low level of BoTx A. (6) In the presence of 4-AP double-poisoning with any combination between BoTx A and TeTx or BoTx B (regardless of intoxication sequence) revealed supra-additive effects, since the number of quanta released was considerably lower than that obtained with any of the toxins alone (in the presence of 4-AP). (7) Our results indicate that tetanus toxin and botulinum toxin type B have a common site of action which is different and independent from that of botulinum toxin type A.

Animals↗
PubMed1987

Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling.

Transient increases of intracellular calcium and exocytotic activity of rat peritoneal mast cells following stimulation with compound 48/80 were monitored using the Ca-indicator dye fura-2 and the capacitance measurement technique. It is known that mast cells very rapidly lose their secretory response towards antigenic or compound 48/80-induced stimulation in the whole-cell recording configuration of the patch-clamp technique due to "washout" of signal mediators. In contrast, we found that calcium transients remained unaffected by intracellular dialysis for as long as 10 min. The fast "washout" phenomenon of exocytosis could be overcome by supplementing the pipette filling solution with guanosinetriphosphate (GTP) indicating a major role for GTP-binding proteins in secretion. The restoration of exocytosis was transient and decayed within three minutes, suggesting diffusional escape of one or several other cytoplasmic substances involved in stimulus-secretion coupling. Quantitative aspects of this process and the implications of its differential effects on Ca-transients versus secretion are discussed.

Animals↗
PubMed1987

The actions of presynaptic snake toxins on membrane currents of mouse motor nerve terminals.

1. The m. triangularis sterni of the mouse was used to investigate the actions of dendrotoxin, beta-bungarotoxin, crotoxin, taipoxin, bee venom phospholipase A2, aprotinin and apamin on presynaptic currents which flow inside the perineural sheath of nerve bundles upon nerve stimulation. 2. Neither the fast K+ current (IK,f) nor the Ca2+-dependent K+ current IK(Ca) (unmasked after blockade of IK,f by 3,4-diaminopyridine) was affected by the neurotoxins and drugs mentioned. 3. Inhibition of both IK,f and IK(Ca) by tetraethylammonium (30 mM) prolonged presynaptic depolarization owing to Ca2+ influx through fast and slow Ca2+ channels. Additional application of dendrotoxin, beta-bungarotoxin, crotoxin or taipoxin in the nanomolar range caused further prolongation of Ca2+ influx, presumably due to blockade of slowly activating K+ current (IK,s). Onset of toxin effects was immediate and could not be reversed by washing for 60 min. 4. Similar prolongation of slow Ca2+ current was effected by 3,4-diaminopyridine, whereas addition of apamin, aprotinin or phospholipase A2 left the signals unchanged. 5. These data indicate that facilitatory actions of dendrotoxin, beta-bungarotoxin, taipoxin and crotoxin are mediated by an increase of Ca2+ entry into nerve terminals. The actions of these toxins are discussed in terms of a blockade of presynaptic K+ channels with slow activation kinetics.

4-Aminopyridine↗
PubMed1986

Beta-bungarotoxin inhibits a non-inactivating potassium current in guinea pig dorsal root ganglion neurones.

beta-Bungarotoxin (beta-BuTx), at concentrations of 0.45-45 nmol/l, selectively reduced a portion of the noninactivating potassium current (IsK) in dorsal root ganglion neurones of the guinea pig, measured by voltage clamp of internally perfused cells. The average reduction of IsK obtainable with beta-BuTx was 34% and usually not completed within 20 min, but irreversible upon washing for 20 min. The I/V-characteristic of the current blocked by beta-BuTx was almost linear. It is suggested that beta-BuTx selectively blocks a noninactivating subtype of potassium channel.

Animals↗
PubMed1986

Two different presynaptic calcium currents in mouse motor nerve terminals.

Extracellular recordings of potential changes under the perineural sheath of nerve bundles close to some of the nerve terminals were performed using the M. triangularis sterni of the mouse. The nerve signals consisted of a predominant double-peaked negativity which was often preceded by a small positive deflection. While the first negative peak is related to the propagating nerve action potential, the second negative deflection can be attributed to a potassium conductance since it was selectively blocked by tetraethylammonium (TEA) or 3,4-diaminopyridine (3,4-DAP). Combined application of TEA and 3,4-DAP gave rise to a prolonged positive-going wave which was blocked by Cd2+, thus, indicating its underlying cause to be a Ca current. Ionophoretic application of TEA and Cd2+ to the endplates affected potassium and calcium components of the subendothelial signals, respectively, thus indicating their presynaptic origin. This finding is supported by the decrease of the amplitude of these components with increasing distance from the endplate region. Maximal effects on K conductance attainable with 3,4-DAP could still be potentiated by TEA, indicating the presence of at least two distinct sets of K channels. The prolonged positive potential induced by TEA and 3,4-DAP consisted of a fast and slow component, both of which can be attributed to Ca conductances with different characteristics. The fast positive signal component is attributed to the voltage-dependent Ca channel, responsible for the initiation of transmitter release. Its amplitude and duration depend on extracellular Ca2+ -concentration. The fast component is still present when Ca2+ is substituted by Sr2+ or Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗
PubMed1986

Dendrotoxin: a selective blocker of a non-inactivating potassium current in guinea-pig dorsal root ganglion neurones.

The voltage clamp technique was used to study the effects of dendrotoxin (DTX) on outward potassium currents in internally perfused dorsal root ganglion neurones of guinea-pig. Sodium currents were eliminated by tetrodotoxin (TTX, 2 mumol/l), calcium currents and calcium-activated potassium conductances were abolished by intracellular perfusion of cells with KF. Depolarizing voltage shifts from a holding potential of -90 mV yielded a fast transient outward current (IfK) and a delayed non-inactivating outward current (IsK). These currents could be separated by shifting the membrane potential to -50 mV, where IfK was almost completely inactivated. DTX, at concentrations of 0.14-1.4 nmol/l selectively reduced a portion of the non-inactivating potassium current, leaving the transient outward current unaffected. Once manifested, the action of DTX could not be reversed by washing. The I-V characteristic of the current blocked by DTX is almost linear and quite different from the one of the 'DTX-resistant' portion of IsK, which shows a non-linear I-V curve. Tetraethylammonium (TEA, 30 mmol/l) strongly reduced IfK and IsK. However, subsequent application of DTX was still able to further reduce IsK. 3,4-diaminopyridine (3,4-DAP, 500 mumol/l) unselectively reduced IfK and a portion of IsK. The remainder of the latter could not further be reduced by DTX, suggesting a similar action of the two blockers on non-inactivating potassium currents. From the results presented, it is suggested that dendrotoxin selectively blocks a non-inactivating subtype of potassium channel.

4-Aminopyridine↗