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Effects of temperature on single calcium channels in canine colonic smooth muscle
1396
ABSTRACTS OF PAPERS
October
EFFECTS OF TEMPERATURE ON SINGLE CALCIUM CHANNELS IN CANINE COLONlC SMOOTH MUSCLE. A. Rich and KM. Sanders Dept. of Physidogy, Univ. of Nevada Schod of Medicine, Reno, NV. 89557. Two components of inward current have been MentWJ in lsdated cdonic smooth muscle cells. One component is transient and reiatively insensitive to dihydropyrldine inhibfiion whereas the second component Is sustained and is more sensitive to dihydropyridines. Previous studies have shown that increasing temperature from 25% to 35% results in a four-fold increase in both components of inward current in these ceils. These studies showed that although nifedipine (1pM) completeiy abdished both current components at 25%. a portion of the transient component is nifedipine-resistant at 35OC. Single channel studies performed at room temperature have revealed a single class of dlhydropyridine-sensitive channels in these ceils. Further experiments were performed to determine if the increase in inward current and the observed nkdipine-resistance at 35% results from 1) an increase in single channel current amplitude, 2) an increase in open probability, and/or 3) the appearance of a dihydropyridine-resistant channel type. Single channel Ba” currents were measured in the cdl-attached configuration. The bath solution contained (in mM): K-aspartate 120, CsCi 20, HEPES 10. and EGTA 5. The pipette sdution contained 80 mM BaCI, and 10 mM HEPES. The figure shows single channei barium currents recorded from a single patch during step depolarizations from 49 mV to -9 mV. increasing temperature from 22% to 32°C resulted in a I.&fold increase in single channei current amplitude and an increase in the open probability. The results suggest that the increase in whde cell inward currents resulting from increasing temperature is due to an increase in both single channel current amplitude and open probability. The appearance of a nifedipine-resistant transient component of inward current may result from an alteration in single channel kinetics rather than the appearance of a dihydropyridine insensitive channel type. (Supported by NIH Grant DK-41315) 2z”c
JPA 32OC
HUMAN
TACHYODDIA
I 40msec
DUE TO OCTREOTIDE.
F.H.Weber, B.Kendall, P.Yeaton, D.Parnbianco, WStevenson, HShaffer, T.Pruett, R.W.McCallum. Univ. of Virginia, Charlottesville, VA. Somatostatin and its analog inhibit the release of many gastrointestinal hormones and have been shown to have potent effects upon duodenal (duo) and gallbladder motor activity. The effect of therapeutic doses of somatostatin analog (octreotide) upon the sphincter of Oddi (SO) has not been investigated in man. Methods: Using a model for human T-tube SO manometry previously described in patients after liver transplantation (Gastro 1992:102 A331), a 6F catheter with side holes located 5,2O,and 22 mm from the distal end was advanced over a .018” guidewire, allowing simultaneous SO and duo manometry. After nonionic contrast cholangiogram, the T-tube was exchanged over a guidewire and the motility catheter advanced into the duodenum. After definition of the SO pressure profile, the 2 proximal side holes were positioned in the high pressure zone of the SO on 3 separate occasions for 7 minutes each time. Data for basal pressure are reported from the last 5 minutes of the 7 minute tracing with the highest basal pressure. Octreotide 100 mcg SC was then administered and SO and duo motor activity recorded for a further 60 minutes. Duo pressure is used as zero baseline and post-octreotide data are reported from the end of the first duo phase Ill activity (mean 15 niinutes after drug was given). Results: Six patients with choiedochocholedochostomv were studied. There was Q significant increase in the mean frequency of phasic SO contractions after octreotide when compared with the basal period (see figure- pc.05). Though the * mean f 1 SD basal pressure increased after octreotide (16.2 + 2.4 vs 19.6 f 3.1 mmHg; p<.O5), changesTa;p&de (75 + 21.6 vs 96.6 f 17.3 mmHg) and duration (4.0 + 0.5 vs 4.7 + 0.4 set) were not significant. Conclusion: Octreotide increases SO basal pressure and SO phasic contractions in man. Further studies should define whether these effects may impair bile flow thereby contributing to gallstone formation in patients receiving chronic therapeutic dosing.
1992
ELECTRICALPROPERTIESOF GALLBLADDER SMOOM MUSCLECELLS. wf, A. Bonev”, M.T. Nelson”, and G.M. Mawet. Depts of tAnat. and Neurobioi.,and oPharmacol.,Univ. of Vermont,Burlington,VT. Numerous studies have employed measures of tension to assess the activity of the gallbladder (GE) muscularis, and the effects of various compoundson GB tone. To determine the electricalevents, and associated ionic currents, that are responsible for GB tone, we have employed intracellular and whole-cell patch clamp recording techniques to study the membrane properties of guinea pig GB smooth muscle cells. In whole-mount preparations, with intracellular microelectrcdes. we found that GB smooth muscle cells were characterized by spontaneous, rhythmic slow wave potentials. These slow waves consisted of: 1) a rapid upstroke depolarization;2) a partial repolarizationfollowed by 3) a sustained plateau potential;and 4) a complete repolarization to RMP. Each slow wave potential was observed to be directly coupled with a contractile twitch of the wholemount preparation. The frequency of slow waves (0.4 f 0.01 Hz) was higher than those that occur throughout the gut tube, and they were of shorter duration (240 4 7 msec). Experiments involving ionic substitution, or compounds that affect selective channels, were done to resolve the currents that underlie the slow wave events. The slow waves could be abolished with Cat+-free Krebs solution, Co (1 mM), or nifedipine (0.1 PM), whereas their amplitude and duration were augmented in presence of Bay K 8644 (500 nM), TEA (5 mM), or 3,4-DAP (2 mM), and by replacing Ca++ with Bat+. The wave form of the slow waves was unaltered by TTX (1.0 PM). Whole-cell patch clamp recordings, from freshly dissociated smooth muscle cells, revealed a transient voltage-gated inward current followed by a sustained voltage-gated outward current. Studies are now being done to characterize the ionic conductances that give rise to these currents. in conclusion, it appears likely that inward flow of Ca++ and outward flow of K+ ions underlie the slow wave potentials in GB smooth muscle cells. It is possible that hormones and neurotransmitters modify GB tone by altering the conductance of these ions, with a resultant change in the frequency and duration of the slow wave potentials. Supported by NS26995.
IMMUNOHISTOCHEMICAL DISTRIBUTION AND CONTRACTILE EFFECIX OF SUBSTANCE P (SP AND CALCITONIN GENERELATED PEPTIDE (CGRP) ON TIL OPOSSUM SPHINCTER OF I, R. De. Gior io S A WyoraI R E. Gleason C Stemini M. Dents. of I&dkn~ and S&ge$, Brigham’ & ‘Women’; &o.$t$ zdycine, I$$~~~~(&~;$. A and CUI=VAMC,
1 x l(ro 2.6 f 0.7 1 x lo* 1.9 f 0.6 1 x IO“ 1.5 f 0.5 Newman-ICeul%&“~< 1 x lti
concentration (#)
BEMlRE 5.0 f 1.1 13.9 f 2.4. 23.6 f .$.‘~&ked 3.0:
1.6 f 0.5 1.6 f 0.5 1.4 f 0.5 to4.4B&F1.2
SP + CGRP 1.3 f 0.4 11.7 f 2.0’ 20.7 f 2.r 26.6 iz 3.8-
ORE (*) & lowest SP
Our data su es~ 1 SP containing nerves are widely &tributed within the opossumg80 an .d’ DUO and many are co-expressed unth CGRP. 2) SPmediated excitation of the in vitro opossum SO may be modulated by CGRF’.
ABSTRACTS OF PAPERS
October
EFFECTS OF TEMPERATURE ON SINGLE CALCIUM CHANNELS IN CANINE COLONlC SMOOTH MUSCLE. A. Rich and KM. Sanders Dept. of Physidogy, Univ. of Nevada Schod of Medicine, Reno, NV. 89557. Two components of inward current have been MentWJ in lsdated cdonic smooth muscle cells. One component is transient and reiatively insensitive to dihydropyrldine inhibfiion whereas the second component Is sustained and is more sensitive to dihydropyridines. Previous studies have shown that increasing temperature from 25% to 35% results in a four-fold increase in both components of inward current in these ceils. These studies showed that although nifedipine (1pM) completeiy abdished both current components at 25%. a portion of the transient component is nifedipine-resistant at 35OC. Single channel studies performed at room temperature have revealed a single class of dlhydropyridine-sensitive channels in these ceils. Further experiments were performed to determine if the increase in inward current and the observed nkdipine-resistance at 35% results from 1) an increase in single channel current amplitude, 2) an increase in open probability, and/or 3) the appearance of a dihydropyridine-resistant channel type. Single channel Ba” currents were measured in the cdl-attached configuration. The bath solution contained (in mM): K-aspartate 120, CsCi 20, HEPES 10. and EGTA 5. The pipette sdution contained 80 mM BaCI, and 10 mM HEPES. The figure shows single channei barium currents recorded from a single patch during step depolarizations from 49 mV to -9 mV. increasing temperature from 22% to 32°C resulted in a I.&fold increase in single channei current amplitude and an increase in the open probability. The results suggest that the increase in whde cell inward currents resulting from increasing temperature is due to an increase in both single channel current amplitude and open probability. The appearance of a nifedipine-resistant transient component of inward current may result from an alteration in single channel kinetics rather than the appearance of a dihydropyridine insensitive channel type. (Supported by NIH Grant DK-41315) 2z”c
JPA 32OC
HUMAN
TACHYODDIA
I 40msec
DUE TO OCTREOTIDE.
F.H.Weber, B.Kendall, P.Yeaton, D.Parnbianco, WStevenson, HShaffer, T.Pruett, R.W.McCallum. Univ. of Virginia, Charlottesville, VA. Somatostatin and its analog inhibit the release of many gastrointestinal hormones and have been shown to have potent effects upon duodenal (duo) and gallbladder motor activity. The effect of therapeutic doses of somatostatin analog (octreotide) upon the sphincter of Oddi (SO) has not been investigated in man. Methods: Using a model for human T-tube SO manometry previously described in patients after liver transplantation (Gastro 1992:102 A331), a 6F catheter with side holes located 5,2O,and 22 mm from the distal end was advanced over a .018” guidewire, allowing simultaneous SO and duo manometry. After nonionic contrast cholangiogram, the T-tube was exchanged over a guidewire and the motility catheter advanced into the duodenum. After definition of the SO pressure profile, the 2 proximal side holes were positioned in the high pressure zone of the SO on 3 separate occasions for 7 minutes each time. Data for basal pressure are reported from the last 5 minutes of the 7 minute tracing with the highest basal pressure. Octreotide 100 mcg SC was then administered and SO and duo motor activity recorded for a further 60 minutes. Duo pressure is used as zero baseline and post-octreotide data are reported from the end of the first duo phase Ill activity (mean 15 niinutes after drug was given). Results: Six patients with choiedochocholedochostomv were studied. There was Q significant increase in the mean frequency of phasic SO contractions after octreotide when compared with the basal period (see figure- pc.05). Though the * mean f 1 SD basal pressure increased after octreotide (16.2 + 2.4 vs 19.6 f 3.1 mmHg; p<.O5), changesTa;p&de (75 + 21.6 vs 96.6 f 17.3 mmHg) and duration (4.0 + 0.5 vs 4.7 + 0.4 set) were not significant. Conclusion: Octreotide increases SO basal pressure and SO phasic contractions in man. Further studies should define whether these effects may impair bile flow thereby contributing to gallstone formation in patients receiving chronic therapeutic dosing.
1992
ELECTRICALPROPERTIESOF GALLBLADDER SMOOM MUSCLECELLS. wf, A. Bonev”, M.T. Nelson”, and G.M. Mawet. Depts of tAnat. and Neurobioi.,and oPharmacol.,Univ. of Vermont,Burlington,VT. Numerous studies have employed measures of tension to assess the activity of the gallbladder (GE) muscularis, and the effects of various compoundson GB tone. To determine the electricalevents, and associated ionic currents, that are responsible for GB tone, we have employed intracellular and whole-cell patch clamp recording techniques to study the membrane properties of guinea pig GB smooth muscle cells. In whole-mount preparations, with intracellular microelectrcdes. we found that GB smooth muscle cells were characterized by spontaneous, rhythmic slow wave potentials. These slow waves consisted of: 1) a rapid upstroke depolarization;2) a partial repolarizationfollowed by 3) a sustained plateau potential;and 4) a complete repolarization to RMP. Each slow wave potential was observed to be directly coupled with a contractile twitch of the wholemount preparation. The frequency of slow waves (0.4 f 0.01 Hz) was higher than those that occur throughout the gut tube, and they were of shorter duration (240 4 7 msec). Experiments involving ionic substitution, or compounds that affect selective channels, were done to resolve the currents that underlie the slow wave events. The slow waves could be abolished with Cat+-free Krebs solution, Co (1 mM), or nifedipine (0.1 PM), whereas their amplitude and duration were augmented in presence of Bay K 8644 (500 nM), TEA (5 mM), or 3,4-DAP (2 mM), and by replacing Ca++ with Bat+. The wave form of the slow waves was unaltered by TTX (1.0 PM). Whole-cell patch clamp recordings, from freshly dissociated smooth muscle cells, revealed a transient voltage-gated inward current followed by a sustained voltage-gated outward current. Studies are now being done to characterize the ionic conductances that give rise to these currents. in conclusion, it appears likely that inward flow of Ca++ and outward flow of K+ ions underlie the slow wave potentials in GB smooth muscle cells. It is possible that hormones and neurotransmitters modify GB tone by altering the conductance of these ions, with a resultant change in the frequency and duration of the slow wave potentials. Supported by NS26995.
IMMUNOHISTOCHEMICAL DISTRIBUTION AND CONTRACTILE EFFECIX OF SUBSTANCE P (SP AND CALCITONIN GENERELATED PEPTIDE (CGRP) ON TIL OPOSSUM SPHINCTER OF I, R. De. Gior io S A WyoraI R E. Gleason C Stemini M. Dents. of I&dkn~ and S&ge$, Brigham’ & ‘Women’; &o.$t$ zdycine, I$$~~~~(&~;$. A and CUI=VAMC,
1 x l(ro 2.6 f 0.7 1 x lo* 1.9 f 0.6 1 x IO“ 1.5 f 0.5 Newman-ICeul%&“~< 1 x lti
concentration (#)
BEMlRE 5.0 f 1.1 13.9 f 2.4. 23.6 f .$.‘~&ked 3.0:
1.6 f 0.5 1.6 f 0.5 1.4 f 0.5 to4.4B&F1.2
SP + CGRP 1.3 f 0.4 11.7 f 2.0’ 20.7 f 2.r 26.6 iz 3.8-
ORE (*) & lowest SP
Our data su es~ 1 SP containing nerves are widely &tributed within the opossumg80 an .d’ DUO and many are co-expressed unth CGRP. 2) SPmediated excitation of the in vitro opossum SO may be modulated by CGRF’.