Effect of Hypercholesterolaemia on Voltage-Operated Calcium Channel Currents in Rabbit Arterial Smooth Muscle Cells

Journal of Human Hypertension (1999) 13, 849–853 1999 Stockton Press. All rights reserved 0950-9240/99 $15.00 http://www.stockton-press.co.uk/jhh Effect of hypercholesterolaemia on voltage-operated calcium channel currents in rabbit arterial smooth muscle cells GF Clunn, S Wijetunge and AD Hughes Clinical Pharmacology, NHLI, St. Mary’s Hospital, Imperial College of Science, Technology and Medicine, South Wharf Road, London, W2 1NY, UK Cholesterol is a major component of cell membranes capacitance was also greater in NZ cells. Consequently, and influences membrane fluidity. Watanabe heritable there was no significant difference in current density hyperpercholesterolaemic rabbits (WHHL) possess between NZ and WHHL cells either in the absence of defective receptors for low density lipoprotein leading drug or in the presence of the calcium channel agonist to increased plasma cholesterol, accumulation of chol- (+)202 791. Current voltage-relationships, kinetics of esterol in the arterial wall and atherosclerosis. In this fast inactivation and steady-state inactivation of IBa also study calcium channel currents (IBa) were compared did not differ significantly between WHHL and NZ. These using conventional whole cell voltage clamp techniques findings suggest that hypercholesterolaemia in WHHL in ear artery cells isolated from control New Zealand has no direct effect on calcium channel current density White rabbits (NZ) with those from WHHL. IBa were larger or voltage-modulation in arterial smooth muscle cells. in cells isolated from NZ than from WHHL, however cell Keywords: calcium channel; cholesterol; vascular smooth muscle; Watanabe hypercholesterolaemic rabbit Introduction atic cholesterol toxicity or other organ damage which develops in cholesterol-fed rabbits.15 WHHL Cholesterol is a major component of cell membranes 1,2 has therefore been proposed to be a model of human and influences membrane structure and fluidity. hereditary hypercholesterolaemia and understand- Changes in membrane fluidity affect the activity of 2 ing pathology in this strain may shed light on a number of enzymes and ion channels, and in human disease processes. addition cholesterol may modulate membrane bound proteins through direct interactions with hydrophobic regions of membrane proteins.3 Materials and methods Incubation with cholesterol has been reported to promote Ca2+ influx into isolated arteries4 and cul- Single arterial smooth muscle cells were freshly dis- 5–8 persed from WHHL and NZ ear arteries by an enzy- tured vascular smooth muscle cells. Voltage- 16 operated calcium channels are a major route of Ca2+ matic digestion technique. Arteries isolated from entry into smooth muscle9 and several studies have WHHL showed no visible evidence of atheromatous implicated this channel in mediating the choles- changes. Vessels from both strains were cut into terol-induced rise in intracellular Ca2+.4,6,10 Elevated short strips (2–3 mm) and incubated in a low- plasma cholesterol may be therefore an important calcium physiological salt solution containing modulator of Ca2+ entry into vascular smooth muscle (mM): NaCl 130, KCl 6, CaCl2 0.01, MgCl2 1.2, glu- and hence influence vascular tone. This action cose 14 and HEPES 10.7 buffered to pH 7.4 with could contribute to the adverse cardiovascular NaOH and 2 mg/ml bovine serum albumin, 1 mg/ml effects of hypercholesterolaemia. We have examined collagenase, 0.5 mg/ml papain and 5 mM dithiothre- itol for 50–60 mins at 37°C. Cells were dispersed by this question further by comparing IBa in single arterial smooth muscle cells isolated from control mild agitation in low-calcium physiological salt sol- New Zealand White rabbits (NZ) with those from ution. After centrifugation the cells were resus- Watanabe heritable hyperpercholesterolaemic strain pended in similar physiological salt solution, but 11 containing 1.7 mM CaCl2. The cells were stored on (WHHL). WHHL rabbits possess defective recep- ° tors for low density lipoprotein (LDL). This results cover slips at 4 C and used within 6 to 8 hours. in a marked elevation of plasma cholesterol,12–14 Experiments were performed using the whole-cell accumulation of cholesterol in the arterial wall12 configuration of the patch-clamp technique with a and marked atherosclerosis,14 but without the hep- List EPC-7 patch-clamp amplifier. Patch pipettes were pulled from borosilicate glass and had resist- ances of 3 to 5M⍀. The junction potential (Ͻ5 mV) Correspondence: Dr AD Hughes, Clinical Pharmacology, NHLI, between the electrode and the bath solution was St. Mary’s Hospital, Imperial College of Science, Technology and subtracted using the Vp offset on the amplifier and Medicine, South Wharf Road, London, W2 1NY, UK electrode capacitance was compensated elec- Hypercholesterolaemia and calcium channels GF Clunn et al 850 tronically. No compensation was made for series inactivating current is half inactivated, k is a slope ⍀ resistance which was less than 5M . The pipette factor and Inon is the fraction of essentially non- 18 solution contained (mM): NaCl 126, MgCl2 1.2, ethy- inactivating current which is present in these cells. ␤ Ј Ј lene glycol-bis ( -amino-ethyl ether) N,N,N ,N - Kinetics of the fast inactivation process of IBa were tetraacetic acid (EGTA) 2, adenosine 5Ј-triphosphate analysed by fitting data to a mono-exponential func- (Mg salt) 2, tetraethylammonium chloride 10, and tion: HEPES 11 buffered to pH 7.2 with NaOH. The − ␶ I = I ·et/ + I experiments were carried out in ‘high-barium sol- t fast non = = ution’ containing (mM): BaCl2 110 and HEPES 10 Where It current at time t, Ifast amplitude of fast- = buffered to pH 7.2 with TEA-OH to increase the size inactivating current, Inon amplitude of non-inactiv- of the inward current elicited by depolarization and ating current and ␶ = time constant of the fast inacti- to minimise calcium-dependent inactivation of cur- vating current. rents.16 Fits were performed by non-linear regression Voltage-clamp command pulses were provided by using Prism 2.01 (GraphPad Software, USA) or cus- a PC via a Labmaster A/D interface board using com- tom macros written for Excel (Microsoft, USA). Data mercially available software (PClamp 5.5, Axon are means ± s.e.m. of n observations. Comparison of Instruments, CA, USA). Data were recorded on-line results was made using a Student’s t-test for after analogue-to-digital conversion at between 2.5– unpaired data. P Ͻ 0.05 was considered significant. 10 kHz depending on the voltage protocol used. The currents were digitally filtered at 2 kHz and leak currents were subtracted digitally using average values Results of steady leakage currents elicited by a 10 mV hyper- Comparison of IBa in NZ and WHHL polarizing pulse. Cell capacitance was estimated as described for fast whole cell recording by Lindau IBa evoked by depolarization from a holding poten- − and Neher,17 assuming a specific membrane capaci- tial of 60 mV were larger in cells isolated from NZ tance of 1 ␮F/cm2. All recordings were made at than from WHHL across a range of activating poten- room temperature. tials (Figure 1a). However cells derived from WHHL had smaller capacitance compared with those derived from NZ (WHHL = 28±2pF, NZ = 40±3pF; Drugs and reagents n = 6 for both; P = 0.02). Thus in terms of current + density there was no significant difference between ( )202791 (isopropyl-4-(2,11,3-benzoxadiazol-4-yl)- the I-V relationship obtained in cells isolated from 1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridine-carbox- the two strains of rabbit (Figure 1b) and voltage- ylate was a gift from Sandoz AG (Basel, dependence of activation did not differ significantly Switzerland). All other chemicals were obtained between the two cell types. Current density in NZ- from Sigma (Dorset, UK). and WHHL-derived cells also did not differ significantly in the presence of a near maximal concen- Statistics and data analysis tration (100 nM) of the dihydropyridine calcium channel agonist, (+)202 791 (Figure 1c). Current-voltage (I–V) relationships were obtained by Steady-state inactivation (hinf) of voltage-operated repeated, progressive depolarization to various test calcium channels was examined by using a 6 sec potentials for 200 ms from a holding potential of pre-conditioning protocol which has been pre- −60 mV. The effect of a drug on I–V relationship was viously shown to induce steady-state inactivation of examined after any response to the drug had stabil- voltage-operated calcium channels in NZ cells.18 ised. The peak inward current at each test potential Normalized hinf relationships were similar in cells was measured. Voltage-dependence of activation from both strains (Figure 1d). was derived from the I–V relationships. I–V data was fitted to a modified Boltzmann function: Kinetics of inactivation (g·(V − Vrev) I = The kinetics of the rapid inactivation of I was mea- (V − Vh) Ba 1 + exp(1 − sured from currents evoked by a 250 ms depolariz- k ation to +20 mV from a holding potential of −60 mV. ␶ where Vrev is the reversal potential, Vh is the poten- The time constant of fast inactivation ( fast) was cal- tial required for half activation g is normalized con- culated from a mono-exponential fit of data as ductance and k is the slope factor. described in Methods assuming an effectively non- Steady-state inactivation data were fitted to a inactivating component of the current, as previously 18 ␶ ± = Boltzmann function: described in NZ cells. fast was 77 19 ms (n 5) in NZ-derived cells and 76 ± 17 ms (n = 5) in WHHL- = Imax derived (NS). I + Inon V − V 1 + expͩ hͪ k Discussion where I is the normalized peak current at any poten- Elevated plasma LDL-cholesterol is a recognised risk 19 tial, Imax is the maximum peak current evoked by factor for cardiovascular disease and atheroscler- such a step, Vh is the holding potential at which osis is reported to increase coronary vasoconstric- Hypercholesterolaemia and calcium channels GF Clunn et al 851 ᭺ ᭹ Figure 1 Current-voltage (I–V) relationships for IBa in ear artery cells derived from WHHL ( ) and NZ ( ).

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