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the myriad of intracellular signaling pathways involving intracellular Ca2+ means Ca2+ plays a role in everything from cellular metabolism to cell death. In heart muscle Ca2+ is primarily known for its role in cellular contraction. Regulation of cardiac muscle contractility is achieved through

Changes in cellular calcium (Ca(2+)) handling are thought to underlie the altered contraction that occurs during cardiac hypertrophy and failure. Recent work has highlighted the importance of t-tubules in the control of intracellular Ca(2+). The present study was performed to investigate whether changes in the distribution of I (Ca) between the surface and t-tubule membranes might contribute to the altered Ca(2+) handling observed during compensated hypertrophy in the spontaneously hypertensive rat (SHR). Experiments were performed on ventricular myocytes isolated from 5-month-old SHR and normotensive Wistar-Kyoto (WKY) control rats. Osmotic shock using formamide was used to disrupt the t-tubular system and the whole-cell patch clamp technique used to monitor I (Ca) in the presence and absence of t-tubules. Membrane capacitance and I (Ca) were greater in control SHR than WKY myocytes; following detubulation, cell capacitance and I (Ca) both decreased and were no longer significantly different in the two cell types. The density of I (Ca) was not significantly different in control SHR and WKY cells or in detubulated myocytes from the two species. These data suggest that the distribution of I (Ca) is unchanged in SHR myocytes compared to WKY controls; I (Ca) density in the t-tubules was 1.2-fold greater than in the sarcolemma in both strains. These data also imply that the increase in surface area in SHR myocytes is due principally to an increase in t-tubular area, which is accompanied by an approximately equivalent increase in I (Ca), so that the density of I (Ca) at the cell surface and in the t-tubules remains the same. These changes would be expected to retain cell function and synchronicity of Ca(2+) release in the SHR at this stage of compensated hypertrophy.

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+from the sarcoplasmic reticulum (SR), and Ni2+(10 mM) was used to inhibit Na+/Ca2+exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+transient amplitude and SR Ca2+content and less Ca2+extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+content and hence Ca2+transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na+load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca2+, originating from an intracellular Ca2+store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243–247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca2+storage pools in the renal diluting segment. Store Ca2+movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca2+was required to maintain store Ca2+content. Removal of either of these substrates resulted in a passive leak of Ca2+from the stores. The uptake of Ca2+into the store was sensitive to the SERCA inhibitor 2,5-di( tert-butyl) hydroquinone, whereas Ca2+release from the store was stimulated by IP3but not cADPR. Store Ca2+was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Δψm, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca2+following exposure to IP3. These results suggest that the endoplasmic reticulum is a dominant Ca2+store in the frog EDT. A second pool, sensitive to ionomycin but not IP3, may overlap with the IP3-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca2+cycling.

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

Sugar beet cells maintained in the stationary phase of the batch culture cycle for 2 or more days have been shown to exhibit many of the characteristics of quiescent (G0) cells. When such cells were subcultured into fresh medium they progressed through a period of DNA synthesis to a highly synchronised first division, 6 days after subculture. The onset of DNA synthesis and cell division were each delayed by 2 days relative to the timing of the events when the cells were subcultured immediately before entry into the stationary phase. The regulation of gene expression during this extended transition from the G0 phase back to the cell division cycle was investigated. The cell division cycle-related genes Bvcdc2, Betvu;CycA2, Arath;CycB1;1 histone H4 and Bvcrk1 (a novel cdc2-like gene) showed widely differing patterns of expression. Bvcdc2 transcripts were present at low levels in quiescent cells whereas crk1, cyclin and histone transcripts were not detectable. Expression of both Bvcrk1 and Betvu; CycA2 was induced within 1 h after subculture into fresh medium, whereas histone H4 gene expression was not detectable for 24 h and showed a marked increase between 24 and 48 h. B-type cyclin transcripts were not detectable until more than 48 h after subculture. The addition of either sucrose or MS macronutrients to quiescent sugar beet cells was not sufficient to re-initiate cell division but both medium components were able to stimulate the expression of the two ‘early’ genes (Betvu;CycA2 and Bvcrk1) within 6 h. Furthermore, although the sugar beet cells were habituated, i.e. they were routinely grown without added plant growth regulators, treatment of quiescent cells with IAA and kinetin also induced expression of Betvu;CycA2 and Bvcrk1 without subsequent cell division.

Sugar beet cells grown in batch suspension culture have been used to study the regulation of polyamine levels during the transition from a quiescent to a proliferating state. The quiescent state was achieved by maintenance of the phytohormone autonomous cells in the stationary phase of the batch culture cycle. After subculture into fresh medium there was an increase in DNA synthesis which was accompanied by a dramatic increase in cellular polyamine levels. The levels of both free and bound cellular putrescine and spermidine within the cells reached a peak before the onset of the first synchronous division. The levels of putrescine, spermidine and to some extent spermine in the culture medium also increased dramatically shortly after subculture. The increase in polyamines was preceded by a rapid but transient increase in omithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50). Arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine synthetase (EC 2.5.1.6) activity did not show the same pattern of cell division-related variation. Inhibition of S-adenosylmethionine biosynthesis with methylglyoxal bis-(guanylhydra-zone) (MGBG) reduced cell division in the suspension culture. Inhibitors of ornithine decarboxylase and arginine decarboxylase individually had little effect on cell division, but in combination led to a reduction in cell division. Addition of polyamines and their precursors to cells in the stationary phase of a batch culture cycle led to the induction of expression of a mitotic cyclin sequence (BvcycII).

Modulation of the L-type Ca2+ channel (LTCC) by sorcin was investigated by measuring the L-type Ca2+ current (I Ca,L) in isolated rabbit ventricular myocytes using ruptured patch, single electrode voltage clamp in the absence of extracellular Na+. Fifty millimolars EGTA (170 nM Ca2+) in the pipette solution buffered bulk cytoplasmic [Ca2+], but retained rapid Ca2+-dependant inactivation of I Ca,L,. Recombinant sorcin (3 μM) in the pipette significantly slowed time-dependant inactivation (τ fast: 8.8 ± 0.9 vs. 15.1 ± 1.7 ms). Sorcin had no significant effect on I Ca,L, after inhibition of the sarcoplasmic reticulum (SR). Using 10 mM 1,2-bis(o-N,N,N′,N′-tetraacetic acid (170 nM Ca2+), I Ca,L inactivation was then determined by a Ca2+ -independent, voltage-dependant process. Under these conditions, 3 μM sorcin speeded up inactivation. A similar effect was observed by substitution of Ca2+ with Ba2+. Down-regulation of endogenous sorcin to 27 ± 7% using an RNAi adenoviral vector slowed inactivation of I Ca,L by ∼42%. The effects of sorcin on voltage-dependant inactivation were mimicked by a truncated form of the protein containing only the Ca2+-binding domain. This data is consistent with two independent actions of sorcin on the LTCC: (1) slowing Ca2+-dependant inactivation and (2) stimulating voltage-dependant inactivation. The net effect of sorcin on the time-dependent inactivation of I Ca,L was a balance between these two effects. Under normal conditions, sorcin slows I Ca,L inactivation because the effects of Ca2+-dependant inactivation out-weigh the effects on voltage-dependant inactivation.

Excitation–contraction (E–C) coupling in cardiac muscle is the transduction mechanism linking the action potential to cell shortening which occurs in response to a transient rise in intracellular calcium (Ca2+i). Activation of the Ca2+ current (ICa,L, the substrate of which is the L-type Ca2+ channel (LTCC) or dihydropyridine receptor (DHPR)) triggers a larger release of Ca2+ from the sarcoplasmic reticulum (SR) store via Ca2+-induced Ca2+ release (CICR) due to activation of the SR Ca2+ release channel, the ryanodine receptor (RyR), on the SR membrane. Experimental evidence strongly supports ‘local control’ of Ca2+ release where a cluster of RyRs are juxtaposed with DHPRs forming a couplon or Ca2+ release unit (CRU) that functions autonomously. The voltage sensitivity of DHPR activity and the progressive recruitment of couplons is the cornerstone of graded Ca2+ release and contractility in cardiac tissue. Although the relationship between trigger Ca2+ and SR Ca2+ release has received much attention, critical questions still remain about the nature of the coupling between LTCC and RyR. In particular, elementary Ca2+ release events (Ca2+ sparks) can be triggered with very high probability during an action potential (AP) but at the peak of the AP the probability of DHPR openings is very much lower than that of Ca2+ sparks (Inoue & Bridge, 2003). Therefore, more than one DHPR must be involved in triggering SR Ca2+ release but it is not clear exactly how many DHPR channels reside in a couplon and how the numbers of channels and their activity modulates SR Ca2+ release. In a recent paper in The Journal of Physiology, Polakova et al. (2008) significantly advance our understanding of the local control process in a complex but powerful study, by demonstrating that a significant number of DHPRs are present in a couplon (20–40) and that at least eight of these channels must be open simultaneously in order to trigger SR Ca2+ release Spontaneous and triggered local release events have been intensively studied since the first description of Ca2+ sparks by Cheng et al. in 1993 (Cheng et al. 1993). An advance on the Ca2+ spark methodology was demonstrated by Song et al. (1998) where a low-affinity Ca2+-sensitive fluorescent probe was combined with high concentrations of EGTA which reported SR Ca2+ release from a couplon. This release activity was termed a Ca2+ spike. Ca2+ spikes are a more appropriate approach where SR Ca2+ release flux is important than measuring Ca2+ sparks, since the Ca2+ spark signal also contains components not directly associated with SR Ca2+ release flux. This is especially evident in the rising and decay phases of Ca2+ sparks, which are intrinsically much slower than Ca2+ spikes. Ca2+ spikes are therefore able to report the latency of Ca2+ release quite faithfully (Zahradnikova et al. 2007). The Ca2+ spike approach involves the use of EGTA in the cell (introduced via the patch pipette), which prevents diffusion of the Ca2+ signal, thereby filtering out global cytosolic changes in Ca2+ whilst the low-affinity indicator reports the large Ca2+ release flux. Polakova and co-workers apply this methodology to rat ventricular myocytes where the Ca2+ spike signal consists of two components: a dominant component attributable to SR Ca2+ release flux, which is manifest as a rapid and transient spike, and a minor component representing the integral of Ca2+ release from the SR. Song et al. (1998) describe this as a ‘pedestal’ since during a voltage pulse this component of the signal remains elevated above baseline. In addition to this powerful methodology, Polakova and colleagues utilize a clever voltage clamp protocol that permits control not only of the number of channels triggered to open but also the driving force acting on those channels. Channels were activated with minimal Ca2+ flux by a depolarizing prepulse to Erev of ICa,L (+60 mV). Rapid repolarization increased the driving force for Ca2+ entry in a temporally synchronized manner and in this aspect the protocol is superior to traditional step depolarizations since channel open probability and driving force would otherwise change in opposite directions. The number of channels activated was controlled by the time spent at +60 mV (1.5 or 5 ms). Line scanning confocal microscopy monitored the occurrence of Ca2+ spikes. A strong component of the research paper was the use of mathematical modelling to describe the stochastic nature of Ca2+ release events (probability, latency) and it is from this computational analysis that many of the significant conclusions of the paper are drawn. The experimental procedure described above was used by the group to demonstrate that the number of DHPRs activated in the couplon is a significant factor in triggering Ca2+ release. Varying the number of channels activated (controlled by the prepulse to +60 mV) affected spike probability and latency. Spike probability was higher and latency was shorter with larger numbers of activated channels. Where maximal numbers of channels were activated compared to only a fraction of channels (5 ms versus 1.5 ms prepulse), the difference between the spike probability was smallest at –40 mV. This repolarization voltage does not inhibit channel reopenings during the tail current since this potential is not sufficiently hyperpolarized to fully inactivate the channels. Therefore, the differences in latency between the two fractions of activated DHPRs were largest at –40 mV since larger numbers of channel reopenings contribute to increasing the synchrony of SR release. Given that these results demonstrate the importance of the number of DHPR channels for SR release, an examination of lengthening the channel open time with the agonist BayK 8644 was performed. This rendered spike properties independent of the repolarization potential, suggesting that, in addition to the numbers of channels open, the channel open time may also be an important factor in the coupling of SR release, a situation that has physiological relevance since it mimics modulation by β-adrenergic agonists in cardiac tissue. However, this result also describes a paradox in the data reported by this group. The analysis carried out by Polakova and colleagues was applied to the distribution of spike latencies. Latency may be interpreted simply as the synchrony of Ca2+ release from the couplon and this synchrony is regulated by the open probability and number of DHPRs in the couplon. As more channels are recruited (5 ms versus 1.5 ms prepulse) synchrony increases, i.e. more spikes occur with shorter latencies. The paradox rests in that prolonging channel openings with BayK 8664, synchrony (latency) was not increased and in fact it was longer than control. The interpretation of these data is that the number of active channels is a dominant feature in the control of SR Ca2+ release flux and not the length of channel openings per se. However, these data are inconsistent with other reports that demonstrate increased synchrony with β-adrenergic stimulation (which also has the effect of increasing channel open times (Song et al. 2001)). Both the application of BayK 8664 and β-adrenergic stimulation shift the voltage dependence of DHPRs in favour of activation at more negative voltages. This may have the effect of (1) recruiting more channels in the couplon which would explain the increased synchrony observed by other groups or (2) the effects of lengthening DHPR open times may not be discernable during an activating prepulse to +60 mV, and explains the paradox reported by the Polakova group. SR load has a significant impact of SR release flux which may also have contributed to this result. The analysis used by Polakova and co-workers enabled description of the coupling fidelity of SR release (i.e. the ability of a single DHPR to trigger SR Ca2+ release). The equation used to explain this includes the number of DHPRs in a couplon, channel open probability and the number of channels that open sequentially (together) (see eqn 4 in the paper). The probability and latency distribution were modelled to derive estimations of these parameters. Significantly, this demonstrated that the coupling fidelity is very low (0.15 at –120 mV) and thus the only way that SR release can occur with high probability (i.e. to mimic the experimental data) is if multiple channels open simultaneously in a couplon. It is from this analysis that the group surmise that at least eight channels must be open in a couplon during activation to trigger SR release and that a couplon contains between 20 and 40 DHPRs. This represents the key advance to our understanding of local control of E–C coupling. The power of this analysis lies in that it quantitatively describes the numbers of DHPRs of a total couplon population that are required to be active in order to trigger SR release and provides a paradigm for explaining the responses to adrenergic stimulation (Song et al. 2001), during pathological remodelling and variable E–C coupling gain. Each of these situations may result in the numbers and activity of DHPRs being modified and explains the capacity for physiological regulation by the local control mechanism. In summary the paper by Polakova and colleagues represents a significant advance in our understanding of the local control mechanism of E–C coupling. The number of channels in a couplon and the minimum number required to open to trigger SR flux are likely to be modified by adrenergic stimulation, regional location, species and during cardiac disease. Application of their approach to these situations is likely to provide a fruitful area for future investigation.

Objective: Recent work has suggested that Na + /Ca 2+ exchange (NCX) and L-type Ca 2+ current (ICa) are located predominantly in the ttubules of cardiac ventricular myocytes, which therefore represent a microdomain for the regulation of intracellular Na + (Nai) and Ca 2+ (Cai). The aim of this study was to investigate the role of the t-tubules in the response of Cai and contraction to interventions that alter the transsarcolemmal Na + gradient. Methods: Enzymatically isolated and detubulated Wistar rat ventricular myocytes were investigated using fluorescence microscopy and optical detection of cell length. Results: In unstimulated cells, spontaneous contractile activity increased when extracellular [Na + ] was decreased or strophanthidin (100 AM) was added to the bathing solution, but the increase was significantly smaller in detubulated cells than in control cells. In electrically stimulated cells, strophanthidin increased Nai to a similar extent in normal and detubulated cells, although the associated increase in Ca 2+ transient amplitude and contraction were significantly smaller in detubulated cells. Similarly, tetrodotoxin (TTX, 10 AM) attenuated the Ca 2+ transient and contraction less in detubulated than in control cells. Increasing stimulation rate (0.05–1 Hz) caused little change or a small increase in contraction amplitude in control cells, but a significant decrease in contraction amplitude in detubulated cells, although the change of Nai caused by increasing stimulation rate from 0 to 1 Hz was not significantly different in the two cells types. Conclusion: It is concluded that although some Na/K ATPase, NCX and Na + channel activity is present on the surface membrane, the t-tubules play a major role in the modulation of contraction via NCX, allowing changes of the transsarcolemmal Na + gradient to be translated into changes of Cai.

A novel gene (RS2) has been isolated from a Beta vulgaris (cv. Regina) cDNA library. The expression of this gene was enhanced in the mature storage organ as compared to leaf tissue. The protein encoded by this gene was found to be alanine- and glutamic acid-rich and it resembles members of the latex allergen Hev b 5 family.

to the editor: The above Viewpoint article summarized and integrated evidence in support of a key role for localized Ca2+ in the regulation of cardiac excitation-contraction (E-C) coupling. The central notion of this is that by virtue of the speed of Ca2+ release and/or the trapping of Ca2+ by