Alexander P. Naumov

Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents.

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca2+]i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca2+]i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na+ and Ca2+ and was blocked by the dihydropyridine Ca2+-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1–5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (−90 mV), and the type II response had inward current that was larger at more positive potentials (−40 to −70 mV). Both types of response were reversible and blocked by removal of extracellular Na+. These results suggest that the rise in [Ca2+]i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na+-dependent conductance, which depolarizes the cell and increases the Ca2+ influx through voltage-gated Ca2+ channels.

Inositol 1,4,5‐trisphosphate activates two types of Ca2+‐permeable channels in human carcinoma cells

Ca2+‐permeable channels in human carcinoma A431 cells were studied using the patch clamp technique. We have found two types of Ca2+‐penneable channels which are activated by inositol 1,4,5‐trisphosphate (IP3) applied to the intracellular side of the plasma membrane. Unitary conductances of these channels are 3.7 and 13 pS (105 mM Ca2+ in recording pipette, 30–33°C). From the extracellular side of the membrane the channels are activated by EGF. It is assumed that extracellular agonists open both channel types by stimulating the release of IP3 from the membrane.

ATP‐activated inward current and calcium‐permeable channels in rat macrophage plasma membranes.

1. To study mechanisms of receptor‐operated Ca2+ influx in non‐excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole‐cell cell‐attached and outside‐out configurations of the patch clamp technique. Under whole‐cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP‐induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4‐. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP‐induced current at ATP4‐ concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady‐state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell‐attached and outside‐out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole‐cell current noise analysis (3.5‐4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current‐voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP‐activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.

ATP-activated Ca2+-permeable channels in rat peritoneal macrophages

The patch‐clamp technique was used to study mechanisms of ATP‐induced Ca2+ influx in rat peritoneal macrophages. The experiments on whole‐cell and patch membranes have shown that extracellular ATP activates channels permeable to di‐ and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca2+, Sr2+, Mn2+, Ba2+ and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non‐hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP‐binding protein is involved in receptor‐to‐channel coupling.

Evidence for involvement of a GTP-binding protein in activation of Ca2+ influx by epidermal growth factor in A431 cells: Effects of fluoride and bacterial toxins

Aluminium fluoride (AlF4-), a G protein activator, was used to study a possible role of G protein in the control of the pathways for Ca2+ influx through plasma membrane of human carcinoma A431 cells. Fluorimetric measurements with the Ca2+ indicator Indo-1 have shown that addition of fluoride induces an increase in concentration of cytosolic free calcium ([Ca2+]in) due to both release of Ca2+ from intracellular stores and Ca2+ influx from the extracellular medium. The cells stimulated by fluoride became unresponsive to subsequent addition of epidermal growth factor (EGF), histamine and bradykinin. The Ca2+ signal induced by fluoride as well as one induced by EGF was inhibited by the pretreatment of cells with protein kinase C activator, phorbol myristate acetate (PMA). The pretreatment of the cells with pertussis toxin produced no effect on EGF-induced calcium response. In contrast, the pretreatment with cholera toxin (CTX) increased the basal level of [Ca2+]in and abolished the effect of EGF. The effects of CTX could not be reproduced by treating the cells with forskolin or IBMX, agents known to elevate cAMP content in the cell. Patch clamp experiments have shown that fluoride increases the activity of Ca(2+)-permeable channels identical to those activated by EGF from the extracellular side of the membrane [Mozhayeva et al. (1991) J. Membr. Biol. 124, 113-126]. The results obtained suggest the involvement of GTP-binding protein in signal transduction from the EGF receptor to Ca(2+)-permeable channel of plasma membrane in A431 cells.

ATP-OPERATED CALCIUM-PERMEABLE CHANNELS ACTIVATED VIA A GUANINE NUCLEOTIDE-DEPENDENT MECHANISM IN RAT MACROPHAGES

1. To elucidate the possible involvement of a G protein in ATP‐evoked Ca(2+)‐permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside‐out and cell‐attached configurations of the patch clamp technique. 2. In inside‐out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/‐ 0.27 (mean +/‐ S.E.M.; n = 16). The delay in the activity development was 21 +/‐ 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S‐evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell‐attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple conductance levels of calcium-permeable channels activated by epidermal growth factor in A431 carcinoma cells

Single Ca(2+)-permeable channels were studied in membrane patches from A431 carcinoma cells. Amplitudes of channel openings fell into three major groups with mean unitary conductances of 1.3, 2.4 and 5.1 pS (105 mM Ca2+ in the pipette as charge carrier). All three groups of events were activated with epidermal growth factor (EGF) from the outside and by GTP non-hydrolyzable analogues from the inside of the patch membrane. As a rule, channel openings were uniform in amplitude in each individual patch but sometimes transitions between openings of different conductance levels were seen. It is concluded that the plasma membrane of A431 cells contains a single type of EGF- and GTP-dependent Ca(2+)-permeable channel (or channel complex) that can display, at least, three conductance levels.

Calcium-permeable channels activated via guanine nucleotide-dependent mechanism in human carcinoma cells

Patch clamp experiments on human carcinoma A431 cells have revealed two types of Ca2+‐permeable channels, the activity of which can be increased by the application of non‐hydrolyzable analogues of GTP to the intracellular side of the membrane. With 105 mM Ca2+ in recording pipette at 30–33°C their unitary conductances (in pS) are 1.3 (SG‐channels) and 2.4 (G‐channels). G‐ and, possibly, SG‐channels are activated from the extracellular side of the membrane with epidermal growth factor (EGF). The data are consistent with the hypothesis that both channels are activated via guanine nucleotide binding (G) proteins.

POTASSIUM CHANNELS IN AORTIC MICROSOMES : CONDUCTANCE, SELECTIVITY, BARIUM-INDUCED BLOCKAGE AND SUBCONDUCTANCE STATES

The activity of potassium channels of canine aortic sarcoplasmic reticulum was measured using the planar lipid bilayer-fusion technique. The channels have a conductance of 208 pS (400/100 mM K+ in cis/trans solutions) and potassium-to-sodium permeability ratio of 7.7 Ba2+ ions produced two main effects: one is the interruption of channel currents for tens to hundreds of milliseconds in a voltage-dependent manner, and the other is the appearance of a second conductance level with amplitude about 60% of the main level.

Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPγS) affects ATP-activated Ca2+-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na+ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+ ∶ Ba2+ ∶ Mn2+ ∶ Sr2+ ∶ Na+ ∶ K+ = 68 ∶ 30 ∶ 26 ∶ 10 ∶ 3.5 ∶ 1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+ ∶ Sr2+ ∶ Na+ ∶ Mn2+ ∶ Ba2+ = 19 ∶ 18 ∶ 14 ∶ 12 ∶ 10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (Kd) of 10 mm. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca2+-permeable channels on the other cell types.