Biomedical subjects

R Z Kozlowski

Publications and source records attributed to R Z Kozlowski.

34 records · Page 2Linked to original sources

PubMed1995

Differential K+ channel distribution in smooth muscle cells isolated from the pulmonary arterial tree of the rat.

Intracellular photorelease of Ca2+ demonstrated the presence of Ca(2+)-activated K+ channels in smooth muscle cells isolated from different locations of the rat pulmonary arterial tree. However, cell-free patch studies revealed marked differences in K+ channel distribution. In the main pulmonary artery the most frequently observed K+ channel was a approximately 245pS conductance Ca(2+)- and ATP-activated (KCa,ATP) channel. In small pulmonary arteries two K+ channel types predominated: the KCa,ATP channel and a approximately 185pS conductance K+ channel insensitive to intracellular Ca2+, ATP and voltage. This difference in K+ channel distribution may highlight a more complex regulatory mechanism for controlling membrane potential in small pulmonary arteries, reflecting their physiologically more important role in governing pulmonary vascular reactivity.

Adenosine Triphosphate↗

PubMed1995

Ca(2+)-activated Cl- and K+ channels and their modulation by endothelin-1 in rat pulmonary arterial smooth muscle cells.

Using the patch-clamp recording technique, we observed that endothelin-1 (ET-1; 0.8-16 nM) enhanced a voltage-activated outward current (Iout) and induced periodic oscillations of inward current in smooth muscle cells isolated from small pulmonary arteries (200-400 microns in diameter). Anion substitution experiments revealed that the ET-1-induced inward current was carried by Cl- ions. Application of bosentan (10 microM; and ETA and ETB receptor antagonist) and FR 139317 (1-10 microM; a selective ETA receptor antagonist) prevented initiation of inward currents or enhancement of Iout by ET-1. The ETB receptor agonist tetra-Ala-endothelin-1 (1-20 nM) failed to evoke these responses. Caffeine (10 mM) induced a single transient inward current and prevented any further activation of inward current, or enhancement of Iout, by subsequent application of 16 nM ET-1, suggesting that these currents were mediated by Ca2+ release from internal stores. Rapid intracellular release of Ca2+ by photolysis of nitr-5 activated an inward Cl- current and increased the magnitude of Iout. These results demonstrate the existence of Ca(2+)-activated Cl- and K+ channels in pulmonary arterial smooth muscle. The physiological role of these channels is at present uncertain, although their activation may be involved in the contractile responses of pulmonary arterial smooth muscle to ET-1.

Adenosine Triphosphate↗

PubMed1995

Ion channels, oxygen sensation and signal transduction in pulmonary arterial smooth muscle.

In contrast to systemic arteries those of the pulmonary circulation constrict in response to hypoxia: a mechanism known as "hypoxic pulmonary vasoconstriction" (HPV). It is has been established that changes in alveolar oxygen tension are the primary stimulus for triggering HPV. The mechanisms underlying this unique response are unknown. Neither the role of the endothelium nor that of the smooth muscle of the pulmonary arteries in oxygen sensing has been elucidated, although both tissues may be involved. Evidence is accumulating to suggest that K+ channels located in the plasma membranes of pulmonary arterial smooth muscle cells may play an important role in oxygen sensing. This process may involve novel mechanisms of signal transduction and K+ channel regulation by charged intermediates, redox reactions or membrane-delimited factors.

Animals↗

PubMed1994

Biophysical properties of Ca(2+)- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat.

A novel class of Ca(2+)-activated K+ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these "KCa,ATP" channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of KCa,ATP channels to voltage, intracellular Ca2+ ([Ca2+]i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K+ and are approximately 20 times more selective for K+ ions than Na+ ions, with a K+ permeability (PK) of 4.6 x 10(-13) cm s-1.Ca2+ ions applied to the intracellular membrane surface of KCa,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca2+ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of KCa,ATP channels by Mg-ATP has an EC50 of approximately 50 microM. Although the EC50 is unaffected by [Ca2+]i, channel activation by Mg-ATP is enhanced by increasing [Ca2+]i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of KCa,ATP channels to Ca2+. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of KCa,ATP channels to Ca2+ and therefore voltage is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.

1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP dependent phosphorylation possibly by interacting with a G-protein coupled directly to the channel. 7. In view of this diverse range of effects, care should be taken when using GTP gamma S to characterize G-protein-mediated events, since the resulting physiological response may be due to activation of several G-protein containing pathways.

Animals↗

PubMed1993

Turnover rate of the cardiac Na(+)-Ca2+ exchanger in guinea-pig ventricular myocytes.

1. Single guinea-pig ventricular myocytes were voltage clamped using the whole-cell configuration of the patch-clamp technique and membrane current generated by the Na(+)-Ca2+ exchange mechanism recorded. 2. Rapid increases in cytosolic free calcium ([Ca2+]i) evoked by flash photolysis of either nitr-5 or DM-nitrophen resulted in current relaxations, arising from a redistribution of exchanger carrier conformations induced by the changes in [Ca2+]i. 3. Relaxation time constants were temperature dependent with a temperature coefficient over a 10 degrees C range (Q10) of approximately 3 and also voltage dependent, decreasing on hyperpolarization for membrane potentials in the range +40 to -80 mV. 4. The experimental results are consistent with consecutive exchange models having electrogenic Na+ translocation steps, together with a site density and turnover rate similar to that for the Na(+)-K+ pump.

Animals↗

PubMed1992

Activation of L-type Ca2+ currents in cardiac myocytes by photoreleased GTP.

L-type calcium currents (ICa) were recorded from isolated ventricular myocytes by using standard patch-clamp methods. In the absence of agonist, photorelease of GTP by flash photolysis of intracellularly applied caged-GTP rapidly increased the amplitude of ICa over a wide range of membrane potentials. Control experiments clearly demonstrated that this effect was not due to either the release of photolytic by-products or to the light flash itself. The timecourse for activation of ICa by photolysis of caged-GTP was markedly altered by intracellular application of either GDP beta S or GTP gamma S. Upon maximal stimulation of ICa by intracellular dialysis with cAMP, photoreleased GTP induced a small, rapid increase in ICa followed by a gradual inhibition. The presence of Rp-cAMPS intracellularly reduced both the magnitude of the response to photoreleased GTP and its time to peak. Similar effects were observed when protein kinase inhibitor dialysed the cell interior, suggesting that both cAMP-dependent and independent processes were involved in this effect. We conclude that rapid release of GTP within ventricular myocytes, in the absence of agonist, causes rapid activation of L-type Ca2+ current. Mechanisms underlying this effect include stimulation of adenylate cyclase, together with other, as yet uncharacterized, GTP-dependent pathways for increasing ICa in the heart.

Animals↗

PubMed1992

Opposing actions of tolbutamide and glibenclamide on hypoxic pulmonary vasoconstriction.

1. We show that cromakalin and diazoxide, drugs that activate ATP-sensitive potassium (KATP) channels, abolish hypoxic pulmonary vasoconstriction (HPV) of isolated, perfused rat lungs. 2. Glibenclamide, an inhibitor of these channels, does not affect HPV, but it reverses the relaxation caused by cromakalim and diazoxide. 3. Tolbutamide, which has effects similar to glibenclamide in other tissues, paradoxically abolishes HPV, an effect reversed by glibenclamide. 4. These results suggest that: (i) pulmonary vessels contain KATP channels which are normally closed and are not opened by levels of hypoxia that cause constriction, (ii) tolbutamide acts on the pulmonary vasculature by a mechanism which differs from that of glibenclamide.

Animals↗

PubMed1992

Nucleotide-dependent activation of KATP channels by diazoxide in CRI-G1 insulin-secreting cells.

1. Patch-clamp recording techniques were used, to examine the effects of diazoxide on KATP currents in CRI-G1 insulin-secreting cells in the presence of non-hydrolysable nucleotides. 2. In the presence of non- or slowly-hydrolyzed ATP analogues, bathing the intracellular aspect of cell-free membrane patches diazoxide inhibited KATP channel activity. 3. Under whole-cell recording conditions, with various non-hydrolysable nucleotides present intracellularly (after dialysis), diazoxide induced KATP current activation. The largest activation occurred with Mg-adenylyl-(beta, gamma-methylene) diphosphate (Mg-AMP-PCP) present in the dialysing solution. This activation was diazoxide- and nucleotide-concentration-dependent. 4. In the absence of Mg2+, or in the presence of manganese (Mn2+) ions intracellularly, diazoxide did not induce KATP current activation, regardless of the species of nucleotide present in the pipette. 5. Intracellularly applied trypsin prevented the activation of KATP currents by diazoxide in the presence of Mg-AMP-PCP, an effect reversed by co-application of intracellular polymethylsulphonyl fluoride with the trypsin. 6. The application, by dialysis, of a CRI-G1 cell lysate, with negligible Mg-ATP, resulted in a substantial activation of the KATP current by diazoxide. 7. It is concluded that diazoxide can activate KATP channel currents by two separate pathways, one requiring a phosphorylation process, the other the presence of an intracellular protein coupled with a Mg-purine nucleotide.

Adenine Nucleotides↗

PubMed1992

Ca2+ and Mg-ATP activated potassium channels from rat pulmonary artery.

We have observed a novel class of calcium-activated potassium channel which is activated by physiological levels of intracellular ATP. These KCa,ATP channels are found on smooth muscle cells isolated from the pulmonary artery. Since their activation by ATP is Mg2+ dependent and is poorly evoked by non- or slowly-hydrolyzed ATP analogues, we conclude that it involves phosphorylation. We suggest that in hypoxia a reduction of intracellular ATP may reduce KCa,ATP channel activity and thereby tend to depolarize the cells. This effect would increase Ca2+ entry through voltage-activated Ca2+ channels and contribute to vasoconstriction.

Adenosine Triphosphate↗

PubMed1991

Voltage-activated currents in the CRI-G1 rat insulin-secreting cell-line.

1. The whole-cell configuration of the patch-clamp recording technique was used to characterize the electrophysiological properties of CRI-G1 insulin-secreting cells. 2. Current-clamp recordings demonstrated the excitable nature of these cells. 3. Voltage-clamp recordings revealed the presence of an inward Na+ current, an inward Ca2+ current and a delayed outward K+ conductance. 4. The electrophysiological properties of CRI-G1 closely resemble those of pancreatic beta-cells, thereby rendering this cell-line as a useful alternative to freshly isolated cells for the study of pancreatic beta-cell electrophysiology and pharmacology.

Action Potentials↗

PubMed1991

Barbiturates inhibit ATP-K+ channels and voltage-activated currents in CRI-G1 insulin-secreting cells.

1. Patch-clamp recording techniques were used to examine the effects of barbiturates upon the ATP-K+ channel, and voltage-activated channels present in the plasma membrane of CRI-G1 insulin-secreting cells. 2. Thiopentone inhibited ATP-K+ channel activity when applied to cell-attached patches or the intracellular or extracellular surface of cell-free patches. Secobarbitone and pentobarbitone were also effective inhibitors of ATP-K+ channels in cell-free patches, whereas phenobarbitone was ineffective. 3. The diabetogenic agent, alloxan, which is structurally related to the barbiturates also produced an inhibition of ATP-K+ channel activity in outside-out patches. 4. Whole-cell ATP-K+ currents were used to quantify the effects of the barbiturates: concentration-inhibition curves for thiopentone, secobarbitone and pentobarbitone resulted in IC50 values of 62, 250 and 360 microM respectively. Phenobarbitone at a concentration of 1 mM was virtually ineffective. 5. Calculation of the apparent membrane concentrations for these drugs indicate that for a given degree of ATP-K+ channel inhibition a similar concentration of each barbiturate is present in the membrane. This suggests that hydrophobicity plays a primary role in their mechanism of action. The pH-dependence and additive nature of barbiturate block also indicates a membrane site of action. 6. Thiopentone, (100 microM) was also found to inhibit differentially voltage-activated whole-cell currents. The relative potency of thiopentone at this concentration was 0.64, 0.38 and 0.12 for inhibiting Ca2+, K+ and Na+ currents respectively when compared with its ability to inhibit the ATP-K+ channel.

Adenosine Triphosphate↗

PubMed1991

Flash photolysis of intracellular caged GTP gamma S increases L-type Ca2+ currents in cardiac myocytes.

L-type calcium currents were recorded from isolated ventricular myocytes using standard patch-clamp methods. Rapid release of photolabile guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), tetralithium salt, by flash photolysis of intracellular caged-GTP gamma S increased the magnitude of the L-type calcium current: an effect independent of the ultraviolet flash per se or the production of photolytic by-products. This increase in calcium current was markedly reduced by intracellularly applied guanosine 5-O-(thiodiphosphate), trilithium salt or by an excess of GTP gamma S. It is therefore likely that rapid release of GTP gamma S intracellularly in the absence of an agonist can, via a G protein-mediated mechanism, cause L-type calcium current activation. In the presence of Rp-adenosine 3,5-mono-thionophosphate (Rp-cAMP), photoreleased GTP gamma S results in a transient and much reduced increase in the amplitude of the L-type calcium current. We conclude that activation of Gs coupled to adenylate cyclase and ultimately cAMP-dependent phosphorylation may be primarily responsible for L-type channel activation, although a fast membrane-delimited (direct) pathway, not involving cytoplasmic second messengers, may also contribute to this effect.

Animals↗

PubMed1990

ATP-sensitive K(+)-channel run-down is Mg2+ dependent.

ATP-sensitive K(+)-channel currents were recorded from isolated membrane patches and voltage-clamped CRI-G1 insulin-secreting cells. Internal Mg2+ ions inhibited ATP-K+ channels by a voltage-dependent block of the channel current and decrease of open-state probability. The run-down of ATP-K+ channel activity was also shown to be [Mg2+]i dependent, being almost abolished in Mg2(+)-free conditions. Substitution of Mn2+ for Mg2+ did not prevent run-down, nor did the presence of phosphate-donating nucleotides, a protease or phosphatase inhibitor or replacement of Cl- by gluconate.

Adenosine Triphosphate↗

PubMed1989

Dual effects of diazoxide on ATP-K+ currents recorded from an insulin-secreting cell line.

1. The effects of diazoxide on ATP-K+ channel currents, recorded from the insulin-secreting cell line, CRI-G1, were studied using patch-clamp techniques. 2. Under current-clamp recording conditions diazoxide (0.6 mM), inhibited action potential activity and hyperpolarized CRI-G1 cells with a concomitant increase in membrane conductance. Recordings from voltage-clamped whole-cells and isolated patches indicate that activation of ATP-K+ channel currents underlie these effects. 3. Diazoxide elicited an activation of ATP-K+ channels which had been partially inhibited by ATP, on application to either surface of the plasma membrane, although it was more effective when applied directly to the cytoplasmic side. Activation of the ATP-K+ currents involves an increase in the single channel open-state probability and an apparent increase in the number of functional channels. 4. Activation was observed only when Mg-ATP was present in the cytoplasmic bathing solution. There was no activation of currents by diazoxide when ATP, in the absence of Mg2+ ions, or Mg-AMP-PNP was present to inhibit the ATP-K+ channels. 5. In the absence of ATP and Mg2+ ions in the cytoplasmic bathing solution, diazoxide (0.6 mM) produced an inhibition of ATP-K+ currents. 6. Cromakalim (BRL 34915) at 10 microM and 100 microM had no significant effects on ATP-K+ currents. 7. It is concluded that diazoxide-induced activation of ATP-K+ channel currents probably involves phosphorylation of the channel or some closely associated membrane protein.

Adenosine Triphosphate↗

PubMed1988

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line.

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.

Adenosine Triphosphate↗