Activation of the hyperpolarization-activated current (if) in sino-atrial node myocytes of the rabbit by vasoactive intestinal peptide

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in extrinsic and intrinsic nerves of the heart. VIP can be released by vagal stimulation but, contrary to ACh, causes positive chronotropic effects as a result of binding to cardiac receptors which stimulate adenylate cyclase, and thus has been implicated in vagal tachycardias. Since the rate of diastolic depolarization of sinoatrial (SA) node myocytes depends on the hyperpolarization-activated current (i_f), which is directly activated by cytoplasmic cAMP, we studied the action of VIP on i f in myocytes isolated from the SA node of the rabbit. VIP (0.65 M) reversibly increased i_f at –65 mV but had no effect at –115 mV suggesting that its primary effect was to shift the activation curve to more positive voltages. Hyperpolarizing ramp and voltage compensation protocols indicated that VIP shifts the activation curve of i_f by approximately 5–6 mV in the positive direction with no change in maximal conductance. This shift may be the mechanism by which VIP produces its positive chronotropic effect and supports a negative feedback role for this peptide during elevated vagal activity.     

Inhibition of the hyperpolarization-activated current (i f) of rabbit SA node myocytes by niflumic acid

The effects of the amphiphilic substance niflumic acid (NFA) were examined in myocytes isolated from the sino-atrial node of the rabbit heart. NFA (50 and 500 μM), for 30–60 s, produced a reversible negative chronotropic effect by reducing the rate of diastolic depolarization, suggesting an inhibitory effect on the hyperpolarization-activated “pacemaker” current (i _f). NFA (from 0.05 to 500 μM) inhibited i _f by modifying the current kinetics, without alteration of the conductance. This was shown by evidence indicating that: (1) NFA inhibited i _f during hyperpolarizing pulses to the mid-point of i _f activation but not at fully activating voltages; (2) the slope and reversal potential of the fully activated current/voltage (I/V) relation were not altered by NFA, indicating no change in slope conductance or ion selectivity; and (3) hyperpolarizing ramp protocols confirmed the lack of action of 50 μM NFA on the fully activated current and a shift of approximately −8 mV. Although similar to inhibition by acetylcholine (ACh), inhibition by NFA was only partly additive with the action of ACh and was not altered by atropine or pertussis toxin, both of which eliminated the action of ACh. The effect of NFA was present after stimulation of adenylate cyclase by forskolin and after inhibition of phosphodiesterase by isobutylmethylxanthine (IBMX). In cell-attached patch measurements, NFA applied externally did not affect i _f measured in the patch. Finally, application of NFA to the cytoplasmic side of excised patches did not alter the current in the absence or presence of adenosine 3′,5′-cyclic monophosphate (cAMP). These results suggest an external, membrane-delimited action of NFA on i _f. Received: 15 June 1995/Received after revision: 26 September 1995/Accepted: 28 September 1995     

Block of the cardiac pacemaker current (If) in the rabbit sino-atrial node and in canine Purkinje fibres by 9-amino-1,2,3,4-tetrahydroacridine

We have investigated the action of 9-amino-1, 2, 3, 4-tetrahydroacridine (THA) on the pacemaker current I _f in rabbit sino-atrial node myocytes and in canine Purkinje fibres. THA at concentrations in the range 3–300 M blocked I _f in a voltage-independent manner, as revealed by measurements on the fully activated I/V relation for I _f. The dose/response relationship of the I _f maximal slope conductance (G_f) can be fitted by assuming a cooperative binding reaction where two THA molecules are required to block one I _f channel. Half-maximal block occurred at 18.2 M in the sino-atrial node and 36.6 M in Purkinje fibres. THA also affected the I _f kinetic properties. This was examined in the sino-atrial node where the current activation curve was shifted in the negative direction on the voltage axis (-21 mV at 30 M THA). The delayed rectifier current, I _k, was also reduced by THA in sino-atrial node myocytes: at –40 mV the I _k fully activated value was decreased to 37% of its control value by 30 M THA, with only a minor modification of the position of the activation curve at the same potential. Thus, although THA blocks I _f at a lower concentration than other known I _f-channel blockers [DiFrancesco (1982) J Physiol (Lond) 329:485-507], its action on the pacemaker current is not specific.     

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (I _Ca,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal I _Ca,L at concentrations in the range 0.05–10 M, without previous -adrenergic stimulation. The ACh-induced reduction of I _Ca,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal I _Ca,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on I _Ca,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.     

Thermodynamic properties of hyperpolarization-activated current (Ih) in a subgroup of primary sensory neurons

Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365–2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33°C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q ₁₀) of 2.9 and 3.1, respectively, while Q ₁₀ of the absolute conductance was 1.3. Using the Arrhenius–Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just ∼2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25°C). Interestingly, the entropy barriers of the slow rates were negative: −15.2R units for activation and −11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (z _g) (3.75 ± 0.32, mean ± SEM, at 25°C) and half-activation potential (V _1/2) (−70.0 ± 1.3 mV at 25°C) did not vary significantly with temperature.     

Niflumic acid reduces the hyperpolarization-activated current (Ih) in rod photoreceptor cells

We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I_h) in newt rod photoreceptors. At 100 μM, NFA delayed the activation of I_h induced by hyperpolarizing voltage pulses to −83 mV from a holding potential of −43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I_h was activated by hyperpolarizing steps to −123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I_h were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I_h tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I_h by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca²⁺ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I_h channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I_f in cardiac myocytes, suggesting that both currents share similar pharmacological properties.     

Mechanism of acetylcholine action on pacemaker current (i f) in canine Purkinje fibers

We have recently reported in canine Purkinje fibers that acetylcholine (ACh) can reverse the positive voltage shift of the pacemaker current (i _f) induced by -adrenergic stimulation while having no direct action of its own [3]. We have now investigated this effect of ACh on the cyclic adenosine monophosphate (cAMP) cascade in more detail. We find that addition of a membrane permeable analogue of cAMP (8-chlorophenylthio cAMP), 0.5–1 mM, increased the amplitude of i _f .This action was not reversed by 1 M ACh, implying that ACh acts at a step prior to cAMP action. We then looked at the steps controlling intracellular concentration of cAMP. Inhibiting the phosphodiesterase with 100 M isobutyl-1-methylxanthine (IBMX) increased i _f. This action, however, was reversed by ACh. Finally we investigated whether the action of forskolin, a direct activator of adenylyl cyclase, could be reversed by ACh. Forskolin (10–20 M) increased i _f, and ACh at 1 M partially reversed this action of forskolin. These results suggest that, in canine Purkinje fibers, ACh reverses the positive action of -adrenergic agents on i _f via a decrease in cAMP production.     

Action of the hyperpolarization-activated current (I h) blocker ZD 7288 in hippocampal CA1 neurons

 The effects of ZD 7288, a ”bradycardic” agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1–1000 μM) reduced the hyperpolarization-activated current (I _h) in CA1 pyramidal neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 μM) caused membrane hyperpolarization (by 5.9 ± 0.5 mV) and a reduction of membrane conductance (by 17.9 ± 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell’s excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs⁺, another known blocker of I _h, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs⁺-induced inhibition of LTD maintenance is not directly related to block of I _h. Received: 14 February 1997 / Received after revision: 4 July 1997 / Accepted: 21 July 1997     

Isolated cells of the frog sinus venosus: properties of the inward current activated during hyperpolarization

Single sinus venosus cells from frog, Rana esculenta, were isolated using an enzymic dispersion procedure, involving applications of collagenase and protease. About 40%–60% of the cells showed spontaneous contractions. Isolated cells were studied in the whole-cell configuration. Regenerative action potentials were tetrodotoxin-insensitive and similar to those recorded in multicellular preparations. Hyperpolarizing pulses in the voltage range negative to –50 mV induced the activation of a time-dependent inward current, which was blocked by 4 mM caesium but less affected by barium ions. A lower concentration of caesium (1 mM) exerted a voltage-dependent reduction of the current and decreased the spontaneous pacing rate. The activation range of the hyperpolarization-activated current approximately extended from –50 mV to –110 mV, but varied from cell to cell. A high variability was observed in the behaviour of the activation kinetics. The current had a reversal potential near –20 mV that was shifted positively by increasing the external potassium concentration (from 3 mM to 30 mM) and negatively by reducing the external sodium concentration (from 115 mM to 30 mM). The hyperpolarization-activated inward current of the frog sinus venosus cell appears to be carried by both sodium and potassium ions. It shows electrophysiological properties similar to those of the I _f current of the mammalian heart. The role of the current in the spontaneous activity is discussed.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

cAMP-dependent inward rectifier current in neurons of the rat suprachiasmatic nucleus

Electrophysiological properties of the inward rectification of neurons in the rat suprachiasmatic nucleus (SCN) were examined by using the single-electrode voltage-clamp method, in vitro. Inward rectifier current (I _H) was produced by hyperpolarizing step command potentials to membrane potentials negative to approximately −60 mV in nominally zero-Ca²⁺ Krebs solution containing tetrodotoxin (1 μM), tetraethylammonium (40 mM), Cd²⁺ (500 μM) and 4-aminopyridine (1 mM).I _H developed during the hyperpolarizing step command potential with a duration of up to 5 s showing no inactivation with time.I _H was selectively blocked by extracellular Cs⁺ (1 mM). The activation of the H-channel conductance (G _H) ranged between −55 and −120 mV. TheG _H was 80–150 pS (n=4) at the half-activation voltage of −84±7 mV (n=4). The reversal potential ofI _H obtained by instantaneous current voltage (I/V) relations was −41±6mV (n=4); it shifted to −51±8mV (n=3) in low-Na⁺ (20 mM) solution and to −24±4 mV (n=4) in high-K⁺ (20 mM) solution. Forskolin (1–10 μM) produced an inward current and increased the amplitude ofI _H. Forskolin did not change the half-activation voltage ofG _H. 8-Bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.1–1 mM) and dibutyryl-cAMP (0.1–1 mM) enhancedI _H. 3-Isobutyl-1-methylxanthine (IBMX, 1 mM) also enhancedI _H. The results suggest that the inward rectifier cation current is regulated by the basal activity of adenylate cyclase in neurons of the rat SCN.     

Regulation of Ca2+ current in frog ventricular myocytes by the holding potential, c-AMP and frequency

The whole-cell patch-clamp technique was used to study the effects of holding potential and frequency on the Ca²⁺ current in frog ventricular myocytes. I _Na was blocked by TTX, and i _ca was activated with depolarizing clamps from different holding potentials. Variation of the holding potential revealed three new effects on i _Ca: (1) At -40 mV i _Ca declined with a time constant of 15 min, while at-90 mV, this irreversible decline (run down) in i _Ca did not occur. (2) The decline of i _Ca at -40 mV was biphasic: run down was preceeded by a slow inactivation with a time constant of 40 s, which was reversible upon returning the holding potential to -90 mV. (3) Increasing the frequency of the clamp pulses from 0.1 to 1 Hz led to a rapid decline of i _Ca when the holding potential was positive to -60 mV, but at -90 mV had either no effect or increased i _Ca by 35%, if c-AMP was included in the dialyzing solution. On the other hand, c-AMP did not alter the time course of the run down and the slow inactivation. Replacement of extracellular Ca²⁺ by Ba²⁺ markedly slowed i _Ca kinetics, but did not change the very slow inactivation or the frequency-induced enhancement of i _Ca. Injection of c-AMP led to a transient increase of i _Ca. The phosphodiesterase inhibitor theophylline enhanced the amplitude of the transient and slowed its decay. This effect was mimicked by increased frequency. It is concluded that frequency-induced enhancement of i _Ca is highly dependent on the holding potential, independent of Ca²⁺, and may involve elevation of the intracellular level of c-AMP via inhibition of phosphodiesterase activity. The new type of very slow inactivation is probably under direct voltage control and independent of Ca²⁺ and c-AMP.     

Noradrenergic modulation of the hyperpolarization-activated cation current (Ih) in dopamine neurons of the ventral tegmental area

Alterations in the state of excitability of midbrain dopamine (DA) neurons from the ventral tegmental area (VTA) may underlie changes in the synaptic plasticity of the mesocorticolimbic system. Here, we investigated norepinephrine's (NE) regulation of VTA DA cell excitability by modulation of the hyperpolarization-activated cation current, Ih, with whole cell recordings in rat brain slices. Current clamp recordings show that NE (40 microM) hyperpolarizes spontaneously firing VTA DA cells (11.23+/-4 mV; n=8). In a voltage clamp, NE (40 microM) induces an outward current (100+/-24 pA; n=8) at -60 mV that reverses at about the Nernst potential for potassium (-106 mV). In addition, NE (40 microM) increases the membrane cord conductance (179+/-42%; n=10) and reduces Ih amplitude (68+/-3% of control at -120 mV; n=10). The noradrenergic alpha-1 antagonist prazosin (40 microM; n=5) or the alpha-2 antagonist yohimbine (40 microM; n=5) did not block NE effects. All NE-evoked events were blocked by the D2 antagonists sulpiride (1 microM) and eticlopride (100 nM) and no significant reduction of Ih took place in the presence of the potassium channel blocker BaCl2 (300 microM). Therefore, it is concluded that NE inhibition of Ih was due to an increase in membrane conductance by a nonspecific activation of D2 receptors that induce an outward potassium current and is not a result of a second messenger system acting on h-channels. The results also suggest that Ih channels are mainly located at dendrites of VTA DA cells and, thus, their inhibition may facilitate the transition from single-spike firing to burst firing and vice versa.     

High selectivity of the i f channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarizationactivated inward current (i _f) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K⁺ pipette, replacement of 90% external Na⁺ by Li⁺ caused a –24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs⁺ pipette, the i _f current decreased almost proportionally to the decrease in external [Na⁺]_o as Li⁺ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG⁺) substitution, suggesting that the relative permeability of Li⁺ compared with Na⁺ for the i _f channel is as low as that of NMG⁺. When Cs⁺ or Rb⁺ was substituted for internal K⁺, the fully activated I/V relationship for i _f showed strong inward rectification with a positive reversal potential, indicating low permeability of the i _f channel for Cs⁺ and Rb⁺. These results show that the i _f channel is highly selective for Na⁺ and K⁺ and will not pass the similar ions Li⁺ and Rb⁺. Such a high degree of selectivity is unique and may imply that the structure of the i _f channel differs greatly from that of other Na⁺ and K⁺ conducting channels.     

Characteristics of the delayed rectifier K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart

The delayed rectifier potassium current (I _K) is known to be important in action potential repolarisation and may contribute to the diastolic pacemaker depolarisation in pacemaker cells from the heart. In this study, using whole-cell patch clamp, we investigated the characteristics of I _K in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at −40 mV and 5 μM external nifedipine was used to block L-type calcium current (I _Ca,L). Significant I _K was observed with pulses to potentials more positive than −30 mV. The steady-state activation curve in both cell types showed maximal activation at between + 10 and + 20 mV. Half-maximal activation of I _K occurred at −4.9 and −4.1 mV with slope factors of 8.3 and 12.4 mV in ventricular and AVN cells, respectively. Using pulses of increasing duration, significant I _K tails after repolarisation from + 40 mV were observed with pulses of 20 ms and increased with pulses up to 100–120 ms in both cell types. Pulses of longer duration did not activate further I _K and this suggested that only the rapid component of I _K, called I _Kr, was present in either cell type. Moreover, I _K tails after pulses to all potentials were blocked completely by E-4031, a selective blocker of I _Kr. The reversal potential of I _K varied with the concentration of external K. Superfusion of AVN cells with medium containing 4, 15 and 40 mM [K⁺]_o resulted in reversal potentials of −81, −56 and −32 mV, respectively, which are close to values predicted if the I _K channel were highly selective for K. The time constants for deactivation of I _K in ventricle and AVN on return to −40 mV after a 500-ms activating pulse to + 60 mV were 480 ms and 230 ms, respectively. The faster deactivation of I _K in AVN cells was a distinguishing feature and suggests that there may be differences in the I _Kr channel protein between ventricular and AVN cells. Received: 24 July 1995 /Received after revision: 20 October 1995 /Accepted: 23 October 1995     

Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells

Studies with apical membrane vesicles have shown that two distinct and separate anion exchange processes are present in rat distal colon, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)-sensitive exchange, and DIDS-resistant Cl⁻–OH⁻ exchange. These studies proposed that anion exchanger (AE)-1 isoform encodes the former as both apical membrane DIDS-sensitive exchange, and AE1 specific mRNA are present only in surface cells and are downregulated in Na-depleted rats, whereas downregulated in adenoma (DRA) encodes the latter as both DIDS-resistant Cl⁻–OH⁻ exchange, and DRA-specific proteins are present in apical membranes of both surface and crypt cells and are not altered in Na⁺-depleted rats. Studies were, therefore, initiated to identify the function of rat DRA (rDRA) in vitro. rDRA cDNA isolated from rat distal colon encodes a 757-amino-acid protein which has 96 and 81% homology with mDRA and hDRA, respectively. rDRA-specific mRNA expression was detectable only in specific segments of the digestive tract (duodenum, ileum, cecum, proximal colon, and distal colon) but not in the stomach, jejunum, or in the kidney, brain, heart, and lung. HEK 293 cells stably transfected with rDRA exhibited DIDS-insensitive and intracellular acid pH (pH_i 6.5)-sensitive Cl uptake that: (1) was significantly stimulated by outward Cl⁻, , isobutyrate, and possibly OH⁻ gradients; (2) was saturated as a function of increasing extracellular Cl concentrations with an apparent K _m for Cl of 2.9 ± 0.3 mM; and (3) was inhibited competitively by extracellular oxalate but not by . A high rate of DIDS-insensitive Cl influx at pH 6.5 was also present under physiological Cl⁻ concentration. Our observations that rDRA mediates DIDS-insensitive, acid pH-dependent Cl⁻ uptake are consistent with prior observations that rDRA does not mediate DIDS-sensitive exchange in rat distal colon. We speculate that, in addition to mediating pH-sensitive Cl⁻ uptake, rDRA may function as a modifier of other anion transport proteins.     

Characteristics of a transient outward current (sensitive to 4-aminopyridine) in Ca2+-tolerant myocytes isolated from the rabbit atrioventricular node

 A transient outward current (I _to) has been observed in the atrioventricular node (AVN), but its characteristics in Ca-tolerant AVN myocytes have not been investigated previously. In this study, I _to was measured from Ca-tolerant rabbit AVN myocytes at 37°C, using the whole-cell patch-clamp technique. With interfering currents inhibited, 500-ms voltage-clamp pulses applied from –80 mV elicited I _to at potentials positive to –30 mV, which increased in magnitude with test potential amplitude. This current was completely blocked by external application of 5 mM 4-aminopyridine (4-AP). During a command pulse, I _to activated rapidly then inactivated with a bi-exponential time-course. Fast and slow time constants of current inactivation (τ_f and τ_s, respectively) showed voltage dependence. At 0 mV, τ_f was 14.5±2.7 ms and τ_s was 112.8±21.2 ms, whilst at +60 mV τ_f was 6.7±1.1 ms and τ_s was 63.7±9.2 ms (n=25). The steady-state inactivation relationship showed half-maximal inactivation at –33.8 mV (n=8). Re-activation of I _to after an inactivating pre-pulse showed a bi-exponential time-course of recovery: τ₁ was 196±70 ms, and τ₂ was 2707±1010 ms (n=6, at –80 mV). Repetitive application of voltage-clamp test pulses showed that I _to inactivation accumulated on repetitive stimulation, but reached a steady state rapidly for a given pulse frequency (0.2–1.0 Hz). AVN I _to was sensitive to the class 1 anti-arrhythmic flecainide (EC₅₀ for peak current of 24 μM), which showed selectivity for the rapidly inactivating current component. Quinidine also inhibited I _to in a dose-dependent fashion, but did not affect the current time-course. Under voltage-clamp conditions, a simulated diastolic depolarisation from –70 to –45 mV did not significantly reduce I _to amplitude, and under current-clamp conditions 4-AP inhibited spontaneous action potentials. Although this is consistent with a significant role for I _to in shaping AVN activity, under the conditions of this study 4-AP also partially blocked the ”rapid” delayed rectifier current, I _Kr, and so the effects of 4-AP on action potentials could not be attributed exclusively to its effects on I _to. Received: 22 October 1998 / Received after revision: 19 January 1999 / Accepted: 20 January 1999     

Serum biochemistry of ostrich (Striothio camelus) in Iran

Serum biochemistry (Striothio camelus), was determined in 75 clinically normal ostrich (42 females and 33 males, 39 under 2 years of age and 36 over 2 years). The following results were obtained: total protein 3.35±0.61 g/dl; prealbumin 0.1±0.01 g/dl; albumin 1.49±0.25 g/dl; α1 globulin 0.24±0.08 g/dl; α2 globulin 0.71±0.19 g/dl; β globulin 0.42±0.18 g/dl; γ globulin 0.63±0.21 g/dl; glucose 163±17 mg/dl; cholesterol 65±15 mg/dl; creatinin 0.26±0.05 mg/dl; triglyceride 151±56 mg/dl; urea 8.68±0.77 mg/dl; uric acid 11.87±3.56 mg/dl; aspartate amino transferase 357±95 U/l; alanin amino transferase activity 14.24±2.7 U/l; alkaline phosphatase 490±241 U/l; and lactate dehydrogenase 1124±31 0U/l.     

Location and nucleotide sequence of the pre-apocytochrome f gene on the Oenothera hookeri plastid chromosome (Euoenothera plastome I)

Summary The gene for pre-apocytochrome f has been mapped by blot hybridization on a 2.4 kbp HindIII fragment of the circular plastid chromosome of Oenothera hookeii employing probes from the corresponding spinach gene. The gene is located distal to the gene for the ATP synthase subunit alpha, at the border of the 45 kbp inversion that distinguishes spinach and Oenothera plastid chromosomes. Both genes are transcribed in the same direction. Nucleotide sequence analysis reveals a single open reading frame encoding 318 amino acids of which 285 comprise the mature polypeptide and another 33 residues represent probably a N-terminal signal sequence. The putative pre-sequence is 2 residues shorter than those known from the spinach, wheat and pea protein. The deduced amino acid sequences of f cytochromes from the four plant species show over 80% conservation, maintaining the structural characteristics of the protein.     

Characterization of basolateral Na/H exchange (Na/H-1) in MDCK cells

MDCK cells were grown to confluent monolayers on permeant filter supports; pH was analysed by using the pH-sensitive fluorescent probe 27-biscarboxyethyl-5,6-carboxyfluorescein and a routine spectrofluorometer equipped with a perfusion cuvette [Krayer-Pawlowska et al. (1990) J Membr Biol 120:173–183]. Superfusion of the basolateral (but not apical) cell surface with Na⁺-containing solutions led to immediate recovery of pH_i from an acid load (NH₄ prepulse). This pH_i recovery was reversibly inhibited by ethylisopropylamiloride indicating Na/H exchange activity. Na/H exchange activity showed an apparent K _m for Na⁺ of about 25 mM Na⁺ and an apparent K _i for inhibition by dimethylamiloride of around 0.2 M; inhibition by dimethylamiloride was competitive with Na⁺ interaction. Lowering pH_i prior to analysis of Na/H exchange leads to sharp activation of Na/H exchange; the apparent V _max for Na/H exchange is increased more than tenfold by lowering the pH_i from 7.0 to 6.7 without an effect on apparent K _m values for Na⁺ interaction. It is concluded that MDCK cells (strain I) grown on a permeant support contain only basolateral Na/H exchange activity, most likely Na/H-1 [for nomenclature see Igarashi et al. (1991) Kidney Int 40:S84–S89].