1. A new preparation of gastric mucosa isolated from new-born piglets is described. The piglet ga... more 1. A new preparation of gastric mucosa isolated from new-born piglets is described. The piglet gastric mucosa was easily separated from the serosal muscle layers by a "blistering" technique which appeared to cause minimal trauma to the tissue and which allowed extended study in vitro in a suitable chamber. Normal resting p.d. was approximately minus 30 mV (mucosal side negative with respect to serosal side), resistance about 100 omega. cm-2 and H+ secretion was absent or occurred at very low rates (0-1mu-equiv/cm-2. hr). 2. Maximally stimulating doses of histamine (1-6 times 10-5 M) caused H+ secretion to increase (up to 15 muequiv/cm-2. hr), p.d. to increase and resistance to decrease. A close correlation was observed between the increase in H+ secretion and decrease in transmucosal resistance. The threshold dose of histamine appeared to be 10-8 M; concentrations 10-4 M and higher reduced H+ secretion somewhat. 3. Pentagastrin ( 10-9-10-7 M) and acetylcholine (10-7-10-5 M) did not significantly stimulate the piglet gastric mucosa. Pentagastrin concentrations above 4 times 10-6 M reversibly inhibited H+ secretion of histamine-stimulated mucosa. High concentrations of acetylcholine (above 4 times 10-4 M) did not affect histamine-stimulated H+ secretion, but a significant reduction in p.d. was observed. 4. This investigation demonstrates the utility of the piglet gastric mucosa for in vitro studies of the mechanism H+ secretion and the action of secretagogues. From a consideration of such factors as the thinness of tissue and ease of preparation it is suggested that neonatal animals may represent a good source of in vitro mammalian gastric tissue.
Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molec... more Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12-triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild-type (WT) and Bax(-/-) /Bak(-/-) (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψmito ), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax-dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψmito and release of cytochrome C and cell death. Re-expression of Bax or Bak in DKO MEF did not alter the WT-like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax-independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψmito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.
American Journal of Physiology-endocrinology and Metabolism, Mar 1, 1978
Gastric mucosas from newborn pigs (0--20 days) and rabbits (0--20 days) were used for in vitro in... more Gastric mucosas from newborn pigs (0--20 days) and rabbits (0--20 days) were used for in vitro investigation of active Na+ transport during resting (no HCl secretion) conditions. As measured with 22Na+, these tissues actively absorb Na+ from the mucosal-to serosal (m-t-s) bathing solution during both open-circuit and short-circuit current (Is) conditions. In the nonsecreting state, net Na+ transport accounts for 40--60% of Isc. The remaining current is provided by net s-to-m flux of Cl-. Amiloride (2-5 X 10(-5) M) in the mucosal solution abolishes this active Na+ transport by inhibiting m-to-s fluxes of Na+ (JNams). In vivo-in vitro experiments showed that active Na+ transport is a normal function of the resting mammalian stomach. Decreasing pH of the mucosal solution below pH 5 reversibly causes decreased current-generating capability of the tissue. Pretreatment of the tissue with amiloride abolishes this pH effect. The implication is that the low pH affects the Na+-entry step into cells. "Titration curves" of current vs. pH had an apparent pK approximately 4.0. Ouabain and K+-free solutions both cause decreases in active Na+ and Cl- current. Calculations indicate that a shunt may account for only a small (less than 30%) percentage of total transepithelial conductance.
Proceedings of the American Philosophical Society: Held at Philadelphia for Promoting Useful Knowledge, Mar 1, 2013
ON 13 JUNE 2010, Richard Darwin Keynes died at the age of ninety. Keynes was a man of enormous en... more ON 13 JUNE 2010, Richard Darwin Keynes died at the age of ninety. Keynes was a man of enormous energy and omniv- orous interests. Born into one of the great intellectual dynas- ties of Cambridge, he was also one of a group of scientists that eluci- dated the mechanism of production of electrical signals in biological tissues. This work established how cells produce the voltage changes that control their function and serve as signals to communicate with other cells. This knowledge is the basis of understanding how the ner- vous system works and it is a scientific contribution as significant as the elucidation of the genetic code. Keynes's broad research interests also led to pioneering work on both nerves and other cells to identify basic physical principles involved in cellular activities. In his later years Keynes became an important editor for several books on Darwin and his travels on the Beagle. Keynes also loved to travel and often found ways to combine his scientific endeavors with collaborations through- out the world, often in South America. Keynes also made significant contributions as a department head and director of a research institute and as an early organizer and proponent of the important field of biophysics.Keynes was born on 14 August 1919, the eldest son of Geoffrey Keynes and Margaret Darwin. Geoffrey Keynes was a very distinguished surgeon at St. Bartholomew's Hospital and at the same time one of the most distinguished English bibliophiles. Geoffrey is best known for his work on Blake, but his work on Donne, Harvey, Gibbon, Austen, and others also has great distinction. When Geoffrey won first prize in the scholarship examination at Barts, Geoffrey's brother, John Maynard, the famous economist responsible for revolutionizing the field of mac- roeconomics and forever altering the policies of governments, wrote to Neville Keynes, their father, "We're really a wonderful family, . . . at ex- aminations. Probably the finest in the kingdom, I expect. If only the examination system lasts, . . . we shall end, I'm sure, by being the Royal Family." Richard's mother was the granddaughter of Charles Darwin. This family relationship had a major impact on Richard's second career as the editor of many of Darwin's documents.Keynes was educated at Oundle School in Peterborough and excelled at science. In 1938 he won a scholarship to Cambridge to study natural sciences, but the outbreak of the Second World War the following year interrupted his studies and also led, upon urging from his uncle, to a re- direction of his studies from medicine to physics. This change proved to be enormously important in that Keynes spent much of his scientific career applying methods and approaches from physics to the study of nerves and other excitable cells, and he also played important adminis- trative roles in establishing the fields of biophysics and physiology.Keynes spent the war years working as an experimental officer, ini- tially for the Anti-Submarine Experimental Establishment, where he was involved with the development of an early form of sonar, and then for the Admiralty Signals Establishment. At the end of the war in 1945, Keynes returned to Cambridge to complete his undergraduate degree. It was at this time that Keynes married Anne Adrian, an accomplished and lovely musician, and daughter of E. H. Adrian, a Nobel laureate and chair of the Physiological Laboratory at Cambridge.Following his undergraduate studies, Keynes completed his Ph.D. thesis under the supervision of Nobel laureate Alan Hodgkin, studying the conduction of electrical impulses along nerve fibers. His wartime work in electronics and engineering turned out to be important, as he designed new devices and was a pioneer in using radioactive tracers (generated in the Cavendish Laboratory cyclotron, built before the war) to study the movement of ions (sodium, potassium, chloride, and cal- cium) into, out of, and within carefully dissected nerves from squids. …
American Journal of Physiology-gastrointestinal and Liver Physiology, Nov 1, 1981
We have tested whether external Ca2+ is required for either initiation or maintenance of secretor... more We have tested whether external Ca2+ is required for either initiation or maintenance of secretory parameters, including membrane elaboration of oxyntic cells, in frog gastric mucosa. Ca2+ was removed from in vitro mucosal preparations [by washing repeatedly in Ca2+-free Ringer solution and adding 0.1 mM ethylene glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid to the serosal solution] either before (i.e., resting tissues) or after addition of stimulants. Electrophysiology [transepithelial potential difference (PD) and resistance], morphology (morphometric analysis of transmission electron micrographs), and transport (H+ secretion) were monitored. La3+ (1 mM) was added to the mucosal solution to help maintain resistance and PD. La3+ decreased tissue shunt conductance during Ca2+-free conditions, as evidenced by a decreased mucosal-serosal flux of 22Na+, presumably by preserving tight-junction integrity. Secretion was elicited by histamine alone or in combination with dibutyryl cAMP and isobutylmethylxanthine (a phosphodiesterase inhibitor). External Ca2+ is not required for the initiation of H+ secretion or the accompanying morphological changes when the combined stimulants are used, whereas H+ secretion and the morphological change showed some Ca2+ dependency when histamine alone was used. Thus, histamine-elicited secretion seems to be more sensitive to Ca2+ removal than that brought about by the combined stimulants. Long-term effects of Ca2+-free solutions on resistance, PD, and H+ secretion can largely be explained by disruptive effects on tight junctions.
American Journal of Physiology-cell Physiology, Apr 1, 1993
The relative Ca transport activities (i.e., of both pumps and leaks) of carbachol-releasable intr... more The relative Ca transport activities (i.e., of both pumps and leaks) of carbachol-releasable intracellular stores and the basolateral plasma membrane of gastric parietal cells were studied using digital image processing of fura-2 fluorescence. Cells were treated with either carbachol (a cholinergic agonist) or thapsigargin (an inhibitor of microsomal Ca-adenosinetriphosphatase) or a combination of the two. Ca-free solutions were used to selectively investigate intracellular store release and plasma membrane pump activity, whereas Ca-containing solutions were used to investigate Ca influx and refilling of the intracellular pool. In the resting cell depletion of the intracellular pool in Ca-free solutions was 15-fold faster than control in the presence of thapsigargin, indicating the efficient (> 90%) recycling of leaked Ca by the store Ca pump. Stimulation with carbachol increased the rate of pool depletion by 70-fold, and this Ca flux out of the internal store was ten times larger than the flux across the plasma membrane. Thus the internal store has ten times greater fluxes (both leaks and pumps) than the plasma membrane during resting and stimulated conditions. After carbachol removal (i.e., reloading) the permeability of the internal store decreases, whereas increased influx across the plasma membrane persists until the store is refilled. Cytoplasmic Ca does not increase during refilling because the intracellular store pump operates eightfold faster than the plasma membrane pump, effectively sequestering Ca as quickly as it enters the cell.
American Journal of Physiology-cell Physiology, Apr 1, 1988
Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of i... more Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of intact rabbit gastric glands loaded with the Ca-sensitive fluorescent dye, fura-2. Cells were repeatedly stimulated with the cholinergic agonist carbachol to gain insights into the membrane mechanisms involved in hormonally stimulated Ca metabolism. In either Ca-containing or Ca-free solutions, carbachol (100 microM) caused a rapid (within 30 s) elevation of Cai from a resting level of 100 nM to greater than 600 nM. After the spike, Cai decreased within 3 min to a lower level that was somewhat elevated (greater than 200 nM) over base line. This plateau was dependent on both carbachol and extracellular Ca (Cao) and could be blocked by the addition of atropine (1 microM) or lanthanum ion (La, 50 microM). The spike is due to the release of Ca from internal stores, whereas the plateau is due to Ca entry across the plasma membrane through agonist-controlled, La-inhibitable channels. After a carbachol stimulation of 3 min or longer, reloading of the internal store was absolutely dependent on Cao. Under these conditions, reloading occurred through a La-sensitive (but nifedipine- and verapamil-insensitive) pathway in the plasma membrane. No significant change in Cai was detectable during the reloading. In contrast to the longer treatments, if carbachol stimulation was terminated with atropine while Cai was still elevated, significant reloading occurred from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
1. A new preparation of gastric mucosa isolated from new-born piglets is described. The piglet ga... more 1. A new preparation of gastric mucosa isolated from new-born piglets is described. The piglet gastric mucosa was easily separated from the serosal muscle layers by a "blistering" technique which appeared to cause minimal trauma to the tissue and which allowed extended study in vitro in a suitable chamber. Normal resting p.d. was approximately minus 30 mV (mucosal side negative with respect to serosal side), resistance about 100 omega. cm-2 and H+ secretion was absent or occurred at very low rates (0-1mu-equiv/cm-2. hr). 2. Maximally stimulating doses of histamine (1-6 times 10-5 M) caused H+ secretion to increase (up to 15 muequiv/cm-2. hr), p.d. to increase and resistance to decrease. A close correlation was observed between the increase in H+ secretion and decrease in transmucosal resistance. The threshold dose of histamine appeared to be 10-8 M; concentrations 10-4 M and higher reduced H+ secretion somewhat. 3. Pentagastrin ( 10-9-10-7 M) and acetylcholine (10-7-10-5 M) did not significantly stimulate the piglet gastric mucosa. Pentagastrin concentrations above 4 times 10-6 M reversibly inhibited H+ secretion of histamine-stimulated mucosa. High concentrations of acetylcholine (above 4 times 10-4 M) did not affect histamine-stimulated H+ secretion, but a significant reduction in p.d. was observed. 4. This investigation demonstrates the utility of the piglet gastric mucosa for in vitro studies of the mechanism H+ secretion and the action of secretagogues. From a consideration of such factors as the thinness of tissue and ease of preparation it is suggested that neonatal animals may represent a good source of in vitro mammalian gastric tissue.
Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molec... more Pseudomonas aeruginosa use N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression in the bacteria. It is expected that in patients with chronic infections with P. aeruginosa, especially as biofilms, local [C12] will be high and, since C12 is lipid soluble, diffuse from the airways into the epithelium and underlying fibroblasts, capillary endothelia and white blood cells. Previous work showed that C12 has multiple effects in human host cells, including activation of apoptosis. The present work tested the involvement of Bak and Bax in C12-triggered apoptosis in mouse embryo fibroblasts (MEF) by comparing MEF isolated from embryos of wild-type (WT) and Bax(-/-) /Bak(-/-) (DKO) mice. In WT MEF C12 rapidly triggered (minutes to 2 h): activation of caspases 3/7 and 8, depolarization of mitochondrial membrane potential (Δψmito ), release of cytochrome C from mitochondria into the cytosol, blebbing of plasma membranes, shrinkage/condensation of cells and nuclei and, subsequently, cell killing. A DKO MEF line that was relatively unaffected by the Bak/Bax-dependent proapoptotic stimulants staurosporine and etoposide responded to C12 similarly to WT MEF: activation of caspase 3/7, depolarization of Δψmito and release of cytochrome C and cell death. Re-expression of Bax or Bak in DKO MEF did not alter the WT-like responses to C12 in DKO MEF. These data showed that C12 triggers novel, rapid proapoptotic Bak/Bax-independent responses that include events commonly associated with activation of both the intrinsic pathway (depolarization of Δψmito and release of cytochrome C from mitochondria into the cytosol) and the extrinsic pathway (activation of caspase 8). Unlike the proapoptotic agonists staurosporine and etoposide that release cytochrome C from mitochondria, C12's effects do not require participation of either Bak or Bax.
American Journal of Physiology-endocrinology and Metabolism, Mar 1, 1978
Gastric mucosas from newborn pigs (0--20 days) and rabbits (0--20 days) were used for in vitro in... more Gastric mucosas from newborn pigs (0--20 days) and rabbits (0--20 days) were used for in vitro investigation of active Na+ transport during resting (no HCl secretion) conditions. As measured with 22Na+, these tissues actively absorb Na+ from the mucosal-to serosal (m-t-s) bathing solution during both open-circuit and short-circuit current (Is) conditions. In the nonsecreting state, net Na+ transport accounts for 40--60% of Isc. The remaining current is provided by net s-to-m flux of Cl-. Amiloride (2-5 X 10(-5) M) in the mucosal solution abolishes this active Na+ transport by inhibiting m-to-s fluxes of Na+ (JNams). In vivo-in vitro experiments showed that active Na+ transport is a normal function of the resting mammalian stomach. Decreasing pH of the mucosal solution below pH 5 reversibly causes decreased current-generating capability of the tissue. Pretreatment of the tissue with amiloride abolishes this pH effect. The implication is that the low pH affects the Na+-entry step into cells. "Titration curves" of current vs. pH had an apparent pK approximately 4.0. Ouabain and K+-free solutions both cause decreases in active Na+ and Cl- current. Calculations indicate that a shunt may account for only a small (less than 30%) percentage of total transepithelial conductance.
Proceedings of the American Philosophical Society: Held at Philadelphia for Promoting Useful Knowledge, Mar 1, 2013
ON 13 JUNE 2010, Richard Darwin Keynes died at the age of ninety. Keynes was a man of enormous en... more ON 13 JUNE 2010, Richard Darwin Keynes died at the age of ninety. Keynes was a man of enormous energy and omniv- orous interests. Born into one of the great intellectual dynas- ties of Cambridge, he was also one of a group of scientists that eluci- dated the mechanism of production of electrical signals in biological tissues. This work established how cells produce the voltage changes that control their function and serve as signals to communicate with other cells. This knowledge is the basis of understanding how the ner- vous system works and it is a scientific contribution as significant as the elucidation of the genetic code. Keynes's broad research interests also led to pioneering work on both nerves and other cells to identify basic physical principles involved in cellular activities. In his later years Keynes became an important editor for several books on Darwin and his travels on the Beagle. Keynes also loved to travel and often found ways to combine his scientific endeavors with collaborations through- out the world, often in South America. Keynes also made significant contributions as a department head and director of a research institute and as an early organizer and proponent of the important field of biophysics.Keynes was born on 14 August 1919, the eldest son of Geoffrey Keynes and Margaret Darwin. Geoffrey Keynes was a very distinguished surgeon at St. Bartholomew's Hospital and at the same time one of the most distinguished English bibliophiles. Geoffrey is best known for his work on Blake, but his work on Donne, Harvey, Gibbon, Austen, and others also has great distinction. When Geoffrey won first prize in the scholarship examination at Barts, Geoffrey's brother, John Maynard, the famous economist responsible for revolutionizing the field of mac- roeconomics and forever altering the policies of governments, wrote to Neville Keynes, their father, "We're really a wonderful family, . . . at ex- aminations. Probably the finest in the kingdom, I expect. If only the examination system lasts, . . . we shall end, I'm sure, by being the Royal Family." Richard's mother was the granddaughter of Charles Darwin. This family relationship had a major impact on Richard's second career as the editor of many of Darwin's documents.Keynes was educated at Oundle School in Peterborough and excelled at science. In 1938 he won a scholarship to Cambridge to study natural sciences, but the outbreak of the Second World War the following year interrupted his studies and also led, upon urging from his uncle, to a re- direction of his studies from medicine to physics. This change proved to be enormously important in that Keynes spent much of his scientific career applying methods and approaches from physics to the study of nerves and other excitable cells, and he also played important adminis- trative roles in establishing the fields of biophysics and physiology.Keynes spent the war years working as an experimental officer, ini- tially for the Anti-Submarine Experimental Establishment, where he was involved with the development of an early form of sonar, and then for the Admiralty Signals Establishment. At the end of the war in 1945, Keynes returned to Cambridge to complete his undergraduate degree. It was at this time that Keynes married Anne Adrian, an accomplished and lovely musician, and daughter of E. H. Adrian, a Nobel laureate and chair of the Physiological Laboratory at Cambridge.Following his undergraduate studies, Keynes completed his Ph.D. thesis under the supervision of Nobel laureate Alan Hodgkin, studying the conduction of electrical impulses along nerve fibers. His wartime work in electronics and engineering turned out to be important, as he designed new devices and was a pioneer in using radioactive tracers (generated in the Cavendish Laboratory cyclotron, built before the war) to study the movement of ions (sodium, potassium, chloride, and cal- cium) into, out of, and within carefully dissected nerves from squids. …
American Journal of Physiology-gastrointestinal and Liver Physiology, Nov 1, 1981
We have tested whether external Ca2+ is required for either initiation or maintenance of secretor... more We have tested whether external Ca2+ is required for either initiation or maintenance of secretory parameters, including membrane elaboration of oxyntic cells, in frog gastric mucosa. Ca2+ was removed from in vitro mucosal preparations [by washing repeatedly in Ca2+-free Ringer solution and adding 0.1 mM ethylene glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid to the serosal solution] either before (i.e., resting tissues) or after addition of stimulants. Electrophysiology [transepithelial potential difference (PD) and resistance], morphology (morphometric analysis of transmission electron micrographs), and transport (H+ secretion) were monitored. La3+ (1 mM) was added to the mucosal solution to help maintain resistance and PD. La3+ decreased tissue shunt conductance during Ca2+-free conditions, as evidenced by a decreased mucosal-serosal flux of 22Na+, presumably by preserving tight-junction integrity. Secretion was elicited by histamine alone or in combination with dibutyryl cAMP and isobutylmethylxanthine (a phosphodiesterase inhibitor). External Ca2+ is not required for the initiation of H+ secretion or the accompanying morphological changes when the combined stimulants are used, whereas H+ secretion and the morphological change showed some Ca2+ dependency when histamine alone was used. Thus, histamine-elicited secretion seems to be more sensitive to Ca2+ removal than that brought about by the combined stimulants. Long-term effects of Ca2+-free solutions on resistance, PD, and H+ secretion can largely be explained by disruptive effects on tight junctions.
American Journal of Physiology-cell Physiology, Apr 1, 1993
The relative Ca transport activities (i.e., of both pumps and leaks) of carbachol-releasable intr... more The relative Ca transport activities (i.e., of both pumps and leaks) of carbachol-releasable intracellular stores and the basolateral plasma membrane of gastric parietal cells were studied using digital image processing of fura-2 fluorescence. Cells were treated with either carbachol (a cholinergic agonist) or thapsigargin (an inhibitor of microsomal Ca-adenosinetriphosphatase) or a combination of the two. Ca-free solutions were used to selectively investigate intracellular store release and plasma membrane pump activity, whereas Ca-containing solutions were used to investigate Ca influx and refilling of the intracellular pool. In the resting cell depletion of the intracellular pool in Ca-free solutions was 15-fold faster than control in the presence of thapsigargin, indicating the efficient (> 90%) recycling of leaked Ca by the store Ca pump. Stimulation with carbachol increased the rate of pool depletion by 70-fold, and this Ca flux out of the internal store was ten times larger than the flux across the plasma membrane. Thus the internal store has ten times greater fluxes (both leaks and pumps) than the plasma membrane during resting and stimulated conditions. After carbachol removal (i.e., reloading) the permeability of the internal store decreases, whereas increased influx across the plasma membrane persists until the store is refilled. Cytoplasmic Ca does not increase during refilling because the intracellular store pump operates eightfold faster than the plasma membrane pump, effectively sequestering Ca as quickly as it enters the cell.
American Journal of Physiology-cell Physiology, Apr 1, 1988
Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of i... more Microspectrofluorimetry was used to measure cytosolic free Ca, Cai, in single parietal cells of intact rabbit gastric glands loaded with the Ca-sensitive fluorescent dye, fura-2. Cells were repeatedly stimulated with the cholinergic agonist carbachol to gain insights into the membrane mechanisms involved in hormonally stimulated Ca metabolism. In either Ca-containing or Ca-free solutions, carbachol (100 microM) caused a rapid (within 30 s) elevation of Cai from a resting level of 100 nM to greater than 600 nM. After the spike, Cai decreased within 3 min to a lower level that was somewhat elevated (greater than 200 nM) over base line. This plateau was dependent on both carbachol and extracellular Ca (Cao) and could be blocked by the addition of atropine (1 microM) or lanthanum ion (La, 50 microM). The spike is due to the release of Ca from internal stores, whereas the plateau is due to Ca entry across the plasma membrane through agonist-controlled, La-inhibitable channels. After a carbachol stimulation of 3 min or longer, reloading of the internal store was absolutely dependent on Cao. Under these conditions, reloading occurred through a La-sensitive (but nifedipine- and verapamil-insensitive) pathway in the plasma membrane. No significant change in Cai was detectable during the reloading. In contrast to the longer treatments, if carbachol stimulation was terminated with atropine while Cai was still elevated, significant reloading occurred from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
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