Characteristics of calcium currents in rabbit portal vein myocytes ROBERT H. COX, DAVID
KATZKA,
Department of Physiology, University The Graduate Hospital, Philadelphia, Cox,
Robert
H.,
David
Katzka,
and
Martin
AND MARTIN of Pennsylvania,
Pennsylvania Morad.
Characteristics
of calcium currents in rabbit portal vein myocytes. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H453-H463, 1992.-The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (&) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ( [Ca2+10), their suppression by organic or inorganic Ca 2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+],). Changing the holding potential from -90 to -40 mV decreased loa from 4.6 t 0.6 to 2.0 t 0.3 pA/pF at 0 mV but did not shift its voltage dependence significantly. The voltage dependence of steady-state inactivation and activation was represented by Boltzmann distributions with the following parameters: inactivation, half-maximal voltage (h.d = -32 t 7 mV and slope factor (k) = 6.1 t 0.2 mV; activation, VOS5= -15 t 4 mV and Fz= 5.6 t 0.6 mV. Doubling the [Ca2+10 increased loa and shifted the voltage dependence of its activation and inactivation by -10 mV toward more positive potentials without altering the window currents. Substituting Na+, Ba2+, or Sr2+ for Ca2+ as the charge carrier through the Ca2+ channel slowed the rate of its inactivation and shifted its voltage dependence toward more negative potentials. Divalent selectivity of the Ca 2+ channel showed an apparent concentration dependence: at 2 mM Is= < lna = &, while at 10 mM ka < Isr = 1na. Because 50-100 PM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid abolished the apparent concentration dependence of the divalent ion selectivity, this phenomenon was attributed to a high Ca2+ selectivity of the channel. Our data support the presence of only one type of Ca2+ channel in rabbit portal vein myocytes with characteristics similar to the L-type Ca 2+ channel described in other cells, but with somewhat different divalent selectivity, holding potential, and [ Na+] 0 dependence. voltage-dependent calcium channels; vascular smooth muscle; L-type channels; ion selectivity; BAY K 8644 CALCIUM CHANNELS play an important role in the function of many types of cells. In vascular smooth muscle they are involved in excitation-contraction coupling (33) and the generation of action potentials (16). Several recent studies have shown that vascular myocytes possess two types of Ca2+ channels (2, 7, 8, 20, 35) similar in characteristics to T- and L-type channels described in other excitable cells (6,22,24). These two channel types differ with respect to their activation and inactivation characteristics, frequency dependence, sensitivity to adrenergic agonists, and selectivity to organic and inorganic Ca2+ channel blockers (22, 24). For the most part, previous studies on vascular myocytes were performed using cells isolated from tonic smooth muscle such as thoracic aorta, ear, tail, and mesenteric arteries. The portal vein of most species, on the other hand, has been characterized as a phasic tissue that exhibits spontaneous action potentials as well as contractions (16). The characteristics of Ca2+ channels 0363-6135/92
$2.00 Copyright
MORAD and Bockus Research Institute,
19146
in this tissue may differ from those of tonic tissues since these slow action potentials are Ca2+ dependent (5). In addition, many but not all (e.g., Ref. 1) previous studies on vascular myocytes have been performed using Ba2+ or high concentrations of Ca2+ as the charge carrier. Because the properties of Ca2* channels may be modified by high concentrations of divalent charge carriers, it was the objective of this study to characterize the biophysical and pharmacological properties of Ca2+ currents in rabbit portal vein using physiological levels of external Ca2+. Our results indicate that rabbit portal vein myocytes possess only one type of Ca2+ channel with characteristics similar to that associated with cardiac L-type Ca2+ channels but with somewhat different ionic selectivity and voltage and external Na+ dependencies. METHODS Tissue and solutions. Single myocytes were enzymatically isolated from portal vein of male New Zealand White rabbits (3-3.5 kg). The portal vein was exposed by midline laparotomy, and the blood vessel was rapidly removed, washed of blood, and dissected free of fat and extraneous tissue in a buffered solution at room temperature. The buffered solution had the following composition (in mM): 140 NaCl, 1 MgC12, 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and IO glucose, with pH 7.4 (titrated with NaOH) and an osmolality of 296 t 2 mosmol/kgH20. The internal solution used to fill the patch pipettes consisted of (in mM) 120 CsCl, 20 tetraethylammonium (TEA) chloride, 5 NaCl, 5 MgATP, IO HEPES, 14 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), and 0.01 adenosine 3’,5’-cyclic monophosphate, with pH 7.2 (titrated with CsOH) and an osmolality of 306 t 3 mosmol/kgH20. Cell isolation procedure. The portal vein was placed in (nominally Ca2+ free) buffer at 37°C for 20 min to remove Ca2+ from the tissue. It was then cut into small pieces (-1 mm2) and placed in 1 ml of cell isolation solution for 20 min at 37°C. The cell isolation solution consisted of incubation solution plus 350 U/ml collagenase (type CLS3; Worthington, Freehold, NJ) and 35 U/ml elastase (porcine pancreas; Calbiochem, La Jolla, CA). The cells and enzyme solution were separated by filtering through 210-pm mesh. The tissue was briefly washed with 1 ml of incubation solution on the mesh, placed in a small beaker with 1.5 ml of incubation solution, and gently aspirated with a Pasteur pipette IO-15 times. Released cells and tissue were then separated by filtering through 210-pm mesh. This process was repeated with 5-min enzyme exposures until the release (usually 2nd or 3rd aspiration) produced cells of good yield and quality as judged by cell appearance with phase-contrast microscopy. An aliquot of cells was transferred to a chamber on the stage of an inverted microscope (Nikon, Diaphot) and allowed to adhere to the chamber’s glass bottom. The chamber was treated weekly with fibronectin (IO pg/ml) to improve cell adhesion. The Ca2+ concentration of the bathing fluid was slowly increased to 2 mM to avoid cell damage.
0 1992 the American
Physiological
Society
H453
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 19, 2019.
H454
cA2+
CHANNELS
IN PORTAL
C
E3 :p-
-20 -10
-\
10
MYOCYTES
surements were repeated at 5 and 10 mM [Ca2+10. [Ca2+10 was then returned to 2 mM, and I-V curves were repeated. If initial and final values of maximum current differed by more than 210%) the data from such cells were rejected. The divalent selectivity of the Ca2+ channels was determined by first recording I-V data from a holding potential of -60 mV with either 2 (n = 4), 5 (n = 12), or 10 mM [Ca2+10 (n = 8). Ba2+ or Sr2+ was then substituted for Ca2+ in random order, and I-V data were obtained with responses to Ca2+ repeated at the end of the sequence. If significant differences (more than &lo%) were found in peak current or voltage at peak current for the I- V curves with Ca2+ between the beginning and end of this sequence, data from that cell were rejected. Tests and evaluations. A number of objective criteria were used to set quality control standards for the cells used in this study. Only elongated cells with smooth tapered ends, smooth gentle curves without sudden changes in width or orientation, and clear appearance were used. Micropipette seals had to be gigaohm in nature with leak currents of
KATZKA,
Department of Physiology, University The Graduate Hospital, Philadelphia, Cox,
Robert
H.,
David
Katzka,
and
Martin
AND MARTIN of Pennsylvania,
Pennsylvania Morad.
Characteristics
of calcium currents in rabbit portal vein myocytes. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H453-H463, 1992.-The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (&) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ( [Ca2+10), their suppression by organic or inorganic Ca 2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+],). Changing the holding potential from -90 to -40 mV decreased loa from 4.6 t 0.6 to 2.0 t 0.3 pA/pF at 0 mV but did not shift its voltage dependence significantly. The voltage dependence of steady-state inactivation and activation was represented by Boltzmann distributions with the following parameters: inactivation, half-maximal voltage (h.d = -32 t 7 mV and slope factor (k) = 6.1 t 0.2 mV; activation, VOS5= -15 t 4 mV and Fz= 5.6 t 0.6 mV. Doubling the [Ca2+10 increased loa and shifted the voltage dependence of its activation and inactivation by -10 mV toward more positive potentials without altering the window currents. Substituting Na+, Ba2+, or Sr2+ for Ca2+ as the charge carrier through the Ca2+ channel slowed the rate of its inactivation and shifted its voltage dependence toward more negative potentials. Divalent selectivity of the Ca 2+ channel showed an apparent concentration dependence: at 2 mM Is= < lna = &, while at 10 mM ka < Isr = 1na. Because 50-100 PM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid abolished the apparent concentration dependence of the divalent ion selectivity, this phenomenon was attributed to a high Ca2+ selectivity of the channel. Our data support the presence of only one type of Ca2+ channel in rabbit portal vein myocytes with characteristics similar to the L-type Ca 2+ channel described in other cells, but with somewhat different divalent selectivity, holding potential, and [ Na+] 0 dependence. voltage-dependent calcium channels; vascular smooth muscle; L-type channels; ion selectivity; BAY K 8644 CALCIUM CHANNELS play an important role in the function of many types of cells. In vascular smooth muscle they are involved in excitation-contraction coupling (33) and the generation of action potentials (16). Several recent studies have shown that vascular myocytes possess two types of Ca2+ channels (2, 7, 8, 20, 35) similar in characteristics to T- and L-type channels described in other excitable cells (6,22,24). These two channel types differ with respect to their activation and inactivation characteristics, frequency dependence, sensitivity to adrenergic agonists, and selectivity to organic and inorganic Ca2+ channel blockers (22, 24). For the most part, previous studies on vascular myocytes were performed using cells isolated from tonic smooth muscle such as thoracic aorta, ear, tail, and mesenteric arteries. The portal vein of most species, on the other hand, has been characterized as a phasic tissue that exhibits spontaneous action potentials as well as contractions (16). The characteristics of Ca2+ channels 0363-6135/92
$2.00 Copyright
MORAD and Bockus Research Institute,
19146
in this tissue may differ from those of tonic tissues since these slow action potentials are Ca2+ dependent (5). In addition, many but not all (e.g., Ref. 1) previous studies on vascular myocytes have been performed using Ba2+ or high concentrations of Ca2+ as the charge carrier. Because the properties of Ca2* channels may be modified by high concentrations of divalent charge carriers, it was the objective of this study to characterize the biophysical and pharmacological properties of Ca2+ currents in rabbit portal vein using physiological levels of external Ca2+. Our results indicate that rabbit portal vein myocytes possess only one type of Ca2+ channel with characteristics similar to that associated with cardiac L-type Ca2+ channels but with somewhat different ionic selectivity and voltage and external Na+ dependencies. METHODS Tissue and solutions. Single myocytes were enzymatically isolated from portal vein of male New Zealand White rabbits (3-3.5 kg). The portal vein was exposed by midline laparotomy, and the blood vessel was rapidly removed, washed of blood, and dissected free of fat and extraneous tissue in a buffered solution at room temperature. The buffered solution had the following composition (in mM): 140 NaCl, 1 MgC12, 10 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and IO glucose, with pH 7.4 (titrated with NaOH) and an osmolality of 296 t 2 mosmol/kgH20. The internal solution used to fill the patch pipettes consisted of (in mM) 120 CsCl, 20 tetraethylammonium (TEA) chloride, 5 NaCl, 5 MgATP, IO HEPES, 14 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), and 0.01 adenosine 3’,5’-cyclic monophosphate, with pH 7.2 (titrated with CsOH) and an osmolality of 306 t 3 mosmol/kgH20. Cell isolation procedure. The portal vein was placed in (nominally Ca2+ free) buffer at 37°C for 20 min to remove Ca2+ from the tissue. It was then cut into small pieces (-1 mm2) and placed in 1 ml of cell isolation solution for 20 min at 37°C. The cell isolation solution consisted of incubation solution plus 350 U/ml collagenase (type CLS3; Worthington, Freehold, NJ) and 35 U/ml elastase (porcine pancreas; Calbiochem, La Jolla, CA). The cells and enzyme solution were separated by filtering through 210-pm mesh. The tissue was briefly washed with 1 ml of incubation solution on the mesh, placed in a small beaker with 1.5 ml of incubation solution, and gently aspirated with a Pasteur pipette IO-15 times. Released cells and tissue were then separated by filtering through 210-pm mesh. This process was repeated with 5-min enzyme exposures until the release (usually 2nd or 3rd aspiration) produced cells of good yield and quality as judged by cell appearance with phase-contrast microscopy. An aliquot of cells was transferred to a chamber on the stage of an inverted microscope (Nikon, Diaphot) and allowed to adhere to the chamber’s glass bottom. The chamber was treated weekly with fibronectin (IO pg/ml) to improve cell adhesion. The Ca2+ concentration of the bathing fluid was slowly increased to 2 mM to avoid cell damage.
0 1992 the American
Physiological
Society
H453
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.215.017.188) on January 19, 2019.
H454
cA2+
CHANNELS
IN PORTAL
C
E3 :p-
-20 -10
-\
10
MYOCYTES
surements were repeated at 5 and 10 mM [Ca2+10. [Ca2+10 was then returned to 2 mM, and I-V curves were repeated. If initial and final values of maximum current differed by more than 210%) the data from such cells were rejected. The divalent selectivity of the Ca2+ channels was determined by first recording I-V data from a holding potential of -60 mV with either 2 (n = 4), 5 (n = 12), or 10 mM [Ca2+10 (n = 8). Ba2+ or Sr2+ was then substituted for Ca2+ in random order, and I-V data were obtained with responses to Ca2+ repeated at the end of the sequence. If significant differences (more than &lo%) were found in peak current or voltage at peak current for the I- V curves with Ca2+ between the beginning and end of this sequence, data from that cell were rejected. Tests and evaluations. A number of objective criteria were used to set quality control standards for the cells used in this study. Only elongated cells with smooth tapered ends, smooth gentle curves without sudden changes in width or orientation, and clear appearance were used. Micropipette seals had to be gigaohm in nature with leak currents of
