Evaluation of an independent prestin mouse model derived from the 129S1 strain.

Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.

Cochlear function in mice with only one copy of the prestin gene.

Targeted deletion of the prestin gene reduces cochlear sensitivity and eliminates both frequency selectivity and outer hair cell (OHC) somatic electromotility. In addition, it has been reported by Liberman and colleagues that F2 generation heterozygotes exhibit a 6 dB reduction in sensitivity, as well as a decrease in protein and electromotility. Considering that the active process is non-linear, a halving of somatic electromotility would be expected to produce a much larger change in sensitivity. We therefore re-evaluated comparisons between heterozygotes and wildtype mice using both in vivo and in vitro electrophysiology, as well as molecular biology. Data reported here for F3-F5 generation mice indicate that compound action potential thresholds and tuning curves, as well as the cochlear microphonic, are similar in heterozygotes and wildtype controls. Measurements of non-linear capacitance in isolated OHCs demonstrate that charge density, as well as the voltage dependence and sensitivity of motor function, is indistinguishable in the two genotypes, as is somatic electromotility. In addition, both immunocytochemistry and western blot analysis in young adult mice suggest that prestin protein in heterozygotes is near normal. In contrast, prestin mRNA is always less than in wildtype mice at all ages tested. Results from F3-F5 generation mice suggest that one copy of the prestin gene is capable of compensating for the deleted copy and that heterozygous mice do not suffer peripheral hearing impairment.

Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1).

Mutations G2370A, G2372A, G2373A, G2375A, Y3937A, S3938A, G3939A and K3940A were made in two potential ATP-binding motifs (amino acids 2370-2375 and 3937-3940) in the Ca(2+)-release channel of skeletal-muscle sarcoplasmic reticulum (ryanodine receptor or RyR1). Activation of [(3)H]ryanodine binding by Ca(2+), caffeine and ATP (adenosine 5'-[beta,gamma-methylene]triphosphate, AMP-PCP) was used as an assay for channel opening, since ryanodine binds only to open channels. Caffeine-sensitivity of channel opening was also assayed by caffeine-induced Ca(2+) release in HEK-293 cells expressing wild-type and mutant channels. Equilibrium [(3)H]ryanodine-binding properties and EC(50) values for Ca(2+) activation of high-affinity [(3)H]ryanodine binding were similar between wild-type RyR1 and mutants. In the presence of 1 mM AMP-PCP, Ca(2+)-activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type and mutants. These observations apparently rule out sequences 2370-2375 and 3937-3940 as ATP-binding motifs. Caffeine or 4-chloro-m-cresol sensitivity, however, was decreased in mutants G2370A, G2373A and G2375A, whereas the other mutants retained normal sensitivity. Amino acids 2370-2375 lie within a sequence (amino acids 2163-2458) in which some eight RyR1 mutations have been associated with malignant hyperthermia and shown to be hypersensitive to caffeine and 4-chloro-m-cresol activation. By contrast, mutants G2370A, G2373A and G2375A are hyposensitive to caffeine and 4-chloro-m-cresol. Thus amino acids 2163-2458 form a regulatory domain (malignant hyperthermia regulatory domain 2) that regulates caffeine and 4-chloro-m-cresol sensitivity of RyR1.

Ryanodine sensitizes the cardiac Ca(2+) release channel (ryanodine receptor isoform 2) to Ca(2+) activation and dissociates as the channel is closed by Ca(2+) depletion.

In single-channel recordings, the rabbit cardiac Ca(2+) release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (P(o)) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = -log(10) Ca concentration). However, at -30 mV, the P(o) was highly sensitive to Ca(2+) added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC(50) value for channel opening was about pCa 8. No significant Ca(2+) inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca(2+) channels is Ca(2+) sensitive, with an EC(50) value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC(50) values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg(2+) decreased the P(o) of ryanodine-modified channels at low Ca(2+) concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P(o) of ryanodine-modified channels. In a [(3)H]ryanodine binding assay, [(3)H]ryanodine dissociation from the high-affinity binding site was found to be Ca(2+) sensitive, with an IC(50) of pCa 7.1. High concentrations of unlabeled ryanodine prevented [(3)H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca(2+) activation of the Ca(2+) release channel and desensitizes Ca(2+) inactivation through an allosteric interaction. [(3)H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca(2+), implying that high-affinity ryanodine and Ca(2+) binding sites are linked through either short- or long-range interactions, probably involving conformational changes.

Functional characterization of mutants in the predicted pore region of the rabbit cardiac muscle Ca(2+) release channel (ryanodine receptor isoform 2).

A highly conserved amino acid sequence, GVRAGGGIGD(4831), which may form part of the Ca(2+) release channel pore in RyR2, was subjected to Ala scanning or Ala to Val mutagenesis; function was then measured by expression in HEK-293 cells, followed by Ca(2+) photometry, high affinity [(3)H]ryanodine binding, and single-channel recording. All mutants except I4829A and I4829T (corresponding to the I4897T central core disease mutant in RyR1) displayed caffeine-induced Ca(2+) release in HEK-293 cells; only mutants G4826A, I4829V, and G4830A retained high affinity [(3)H]ryanodine binding; and single-channel function was found for all mutants tested, except for G4822A and A4825V. EC(50) values for caffeine-induced Ca(2+) release were increased for G4822A, R4824A, G4826A, G4828A, and D4831A; decreased for V4823A; and unchanged for A4825V, G4827A, I4829V, and G4830A. Ryanodine (10 microm), which did not stimulate Ca(2+) release in wild type (wt), did so in Ala mutants in amino acids 4823-4827. It inhibited the caffeine response in wt and most mutants, but enhanced the amplitude of caffeine-induced Ca(2+) release in mutant G4828A. It also restored caffeine-induced Ca(2+) release in mutants I4829A and I4829T. In single-channel recordings, mutants I4829V and G4830A retained normal conductance, whereas all others had decreased unitary channel conductances ranging from 27 to 540 picosiemens. Single-channel modulation was retained in G4826A, I4829V, and G4830A, but was lost in other mutants. In contrast to wt and G4826A, I4829V, and G4830A, in which divalent metals were preferentially conducted, mutants with loss of modulation had no selectivity of divalent cations over a monovalent cation. Analysis of Gly(4822) to Asp(4831) mutants in RyR2 supports the view that this highly conserved sequence constitutes part of the ion-conducting pore of the Ca(2+) release channel and plays a key role in ryanodine and caffeine binding and activation.

Mutual up-regulation of thyroid hormone and parathyroid hormone receptors in rat osteoblastic osteosarcoma 17/2.8 cells.

PTH and thyroid hormone (T(3)) stimulate anabolic and catabolic processes in bone predominantly by acting on osteoblasts. Both inadequate and excessive secretion of either hormone can result in clinical bone disorders. In addition, T(3) and PTH related peptide (PTHrP) have multiple effects on a wide number of other tissues modulating both cell differentiation and proliferation. To address the question of whether there might be functional mutual regulation of T(3) receptors (TR) and PTH/PTHrP receptors (PTHR), we studied their expression and receptor-mediated intracellular effects in rat osteoblastic osteosarcoma (ROS) 17/2.8 cells. PTHR were up-regulated by T(3) pretreatment (10(-)(10)-10(-)(6) M) in ROS 17/2.8 cells in a dose-dependent manner. T(3) pretreatment increased both PTH-induced cyclic AMP response element binding protein (CREB) phosphorylation and PTH-induced intracellular calcium transients, and further decreased PTH-induced down-regulation of alkaline phosphatase activity, suggesting that there are functional consequences of the PTHR up- regulation. Pretreatment with PTH (10(-)(10)-10(-)(6) M) or PTHrP (10(-)(9) M) for 3-4 days resulted in a dose-dependent up-regulation of TR in ROS 17/2.8 cells. cAMP analogues or a calcium ionophore were able to mimic the effect of PTH on TR binding, suggesting that either the cAMP-signaling pathway or Ca(2+) could be involved in PTH-induced up-regulation of the TR. These observations provide a novel example of mutual interactions between nuclear receptors and membrane receptors and may have significant implications for the regulation of bone remodeling in health and disease.

Mutation of divergent region 1 alters caffeine and Ca(2+) sensitivity of the skeletal muscle Ca(2+) release channel (ryanodine receptor).

Replacement of amino acids 4187-4628 in the skeletal muscle Ca(2+) release channel (skeletal ryanodine receptor (RyR1)), including nearly all of divergent region 1 (amino acids 4254-4631), with the corresponding cardiac ryanodine receptor (RyR2) sequence leads to increased sensitivity of channel activation by caffeine and Ca(2+) and to decreased sensitivity of channel inactivation by elevated Ca(2+) (Du, G. G., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 26120-26126). In further investigations, this region was subdivided by the construction of new chimeras, and alterations in channel function were detected by measurement of the caffeine dependence of in vivo Ca(2+) release and the Ca(2+) dependence of [(3)H]ryanodine binding. Chimera RF10a (amino acids 4187-4381) had a lower EC(50) value for activation by caffeine, and RF10c (4557-4628) had a higher EC(50) value, whereas the EC(50) value for chimera RF10b (4382-4556) was unchanged. Chimeras RF10b and RF10c were more sensitive to activation by Ca(2+), whereas RF10a was less sensitive to inactivation by Ca(2+), implicating RF10b and RF10c in Ca(2+) activation and RF10a in Ca(2+) inactivation. Deletion of much of divergent region 1 sequence to create mutant Delta4274-4535 led to higher caffeine and Ca(2+) sensitivity of channel activation and to lower Ca(2+) sensitivity for inactivation. Thus, deletion results demonstrate that caffeine, Ca(2+), and ryanodine binding sites are not located in amino acids 4274-4535. Nevertheless, the properties of the deletion and chimeric mutants demonstrate that amino acids 4274-4535 and three shorter sequences in this region (F10a, amino acids 4187-4381; F10b, 4382-4556; and F10c, 4557-4628) in RyR1 modulate Ca(2+) and caffeine sensitivity of the Ca(2+) release channel.

HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine.

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca(2+) homoeostasis, the properties of intracellular Ca(2+) stores and the methods used for measuring intracellular Ca(2+) release in HEK-293 cells were evaluated. Ca(2+) imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca(2+) release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca(2+) release in untransfected HEK-293 cells. Intracellular Ca(2+) release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca(2+) release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [(3)H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

Ca(2+) inactivation sites are located in the COOH-terminal quarter of recombinant rabbit skeletal muscle Ca(2+) release channels (ryanodine receptors).

Ca(2+) activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca(2+) release channels (ryanodine receptors) occurs with EC(50) values of about 1 microM. Ca(2+) inactivation occurs with an IC(50) value of about 3.7 mM for RyR1, but RyR2 shows little inactivation, even at >100 mM Ca(2+). In an attempt to localize the low affinity Ca(2+) binding sites responsible for Ca(2+) inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [(3)H]ryanodine binds only to open channels, and because channel opening and closing are Ca(2+)-dependent, the Ca(2+) dependence of [(3)H]ryanodine binding was used as an indirect measurement of Ca(2+) release channel opening and closing. IC(50) values for [(3)H]ryanodine binding suggested that Ca(2+) affinity for the low affinity Ca(2+) inactivation sites was unchanged in a chimera in which a glutamate-rich sequence (amino acids 1743-1964) in RyR1 was replaced with the corresponding, less acidic sequence from RyR2. Ca(2+) affinity (IC(50)) for low affinity Ca(2+) inactivation sites was intermediate in RyR1/RyR2 chimeras containing RyR2 amino acids 3726-4186 (RF9), 4187-4628 (RF10), or 4629-5037 (RF11), was closer to RyR2 values in RyR1 chimeras with longer RyR2 replacements (RF9/10 or RF10/11), and was indistinguishable from RyR2 in RyR1 containing all three RyR2 replacements (RF9/10/11). These data suggest that multiple low affinity Ca(2+) binding sites or multiple components of a low affinity Ca(2+) binding site are located between amino acids 3726 and 5037 and that their effects on Ca(2+) inactivation of the release channel are cooperative. Measurement of Ca(2+) activation of [(3)H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca(2+) than was either RyR1 or RyR2. Measurement of caffeine activation of Ca(2+) release in vivo showed that chimeras RF9, RF10, RF9/10, RF10/11, and RF9/10/11 were more sensitive to caffeine than wild-type RyR1. These results suggest that Ca(2+) and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2.

Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors.

The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor (EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGF-induced receptor down-regulation. Furthermore, the over-expression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma SMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/time-dependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.

Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay.

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres.

We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)2 the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of -0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca2+)/P(K+)=2.4, P(Ca2+)/ P(Na+)=2.7, P(Ca2+)/P(Tris+)=3.1, P(Ca2+)/P(Mg2+)=1.0 and P(Ca2+)/P(Ba2+)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)2 solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)2 solution. The channel activity in symmetrical 50 mM Ca(gluconate)2 solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 microM). The results are in agreement with the conclusion that the sarcoball Ca2+ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca2+-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel.

[Regulatory effect of hepatic stimulator substance on the proliferation of human hepatoma cells].

The hepatic stimulator substance (HSS) was prepared from weanling rat livers by ultracentrifugation and Sephadex G-75 chromatography. The stimulatory effects of HSS on cell proliferation, EGF receptor expression and receptor tyrosine phosphorylation were investigated in serum-free cultured human hepatoma cell line SMMC-7721. The results showed: (1) The relative percentage of S-phase cells was increased in HSS-treated cells. (2) EGF receptor protein level was increased after the cells were treated with HSS for 12-24 h and this effect was dose- and time-dependent. (3) Treatment of the cells with HSS for 0.5-15 min resulted in the autophosphorylation of EGF receptor tyrosine residue dramatically. The above results suggest that the proliferative action of HSS on hepatocytes might be due to its modulation of EGF receptor expression and the signaling pathway.

Ca2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: effects of cyclopiazonic acid and thapsigargin.

Ca2+ efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca2+ in the presence of ATP and inorganic phosphate (Pi). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM Pi, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM Pi. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 microM, which completely inhibit Ca2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6-7 nmole/mg/min was recorded. TG (2.5 microM) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca(2+)-ATPase passive channel.

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake, spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae.

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca2+ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca2+ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca2+ content loaded into the SR, (2) the spontaneous Ca2+ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca2+ sensitivity in Triton X-100-treated preparations. Inhibition of Ca2+ loading by TG and CPA increased with time of exposure to the inhibitor over 18-24 min. TG and CPA produced half inhibition of Ca2+ loading at 34.9 and 35.7 microM respectively, when 18-24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 microM TG and 30 microM CPA both abolished the oscillations in this time. The myofilament Ca2+ sensitivity was not affected by 10 and 300 microM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca2+ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation.

The thyrotropin (TSH) receptor transmembrane domain mutation (Pro556-Leu) in the hypothyroid hyt/hyt mouse results in plasma membrane targeting but defective TSH binding.

The hyt/hyt mouse is hypothyroid because of a mutation in the TSH receptor (TSH-R). In this report, we confirm the presence of a Pro to Leu mutation in amino acid 556 of the fourth transmembrane domain (TM4) of the TSH-R. This Pro is highly conserved in members of the G protein-coupled seven-transmembrane family of receptors. Insertion of this mutation into the wild-type rat receptor eliminated TSH binding and receptor function in transfected 293 and COS cells. Wild-type TSH-R conferred a 7.4-fold increase in cAMP and a 2.3-fold stimulation of a cAMP-responsive reporter gene. The P556L mutant receptor elicited no increase in cAMP or the reporter gene. Cells transfected with wild-type receptor bound TSH with a Kd of 3.3 x 10(-10) M, whereas no TSH binding was detected with the P556L mutant. Because the P556L mutation occurs in a receptor region (TM4) that is not expected to alter the binding of TSH, additional studies were performed to examine receptor processing and cellular localization. Mutant receptors from solubilized membranes also failed to bind TSH, indicating that the absence of binding to intact cells was not accounted for intracellular trapping of the mutant receptor. Western blot analyses demonstrated that the mutant and wild-type receptors were processed through a similar series of precursors and that a mature 95-kilodalton form of the mutant TSH-R was produced, consistent with its insertion into the plasma membrane. Immunofluorescence studies confirmed expression of the P556L mutant on the cell surface of transfected cells and in thyroid tissue from hyt/hyt mice. Although the extracellular domain of the TSH-R is sufficient for high affinity binding of TSH, we conclude that the hyt mutation in the fourth transmembrane domain eliminates TSH binding. These results suggest interactions between the extracellular and transmembrane domains of the TSH-R and indicate that this highly conserved proline is required for normal receptor structure and function.

Helicobacter pylori associated with a high prevalence of duodenal ulcer disease and a low prevalence of gastric cancer in a developing nation.

This study examines the relationship between Helicobacter pylori infection and peptic ulcer disease and gastric cancer--in particular, the presence or absence of bacteria, the grading of gastritis, and the degree of inflammation in the antral and oxyntic mucosae. The grading of gastritis and the detection of H pylori were determined by histology using the Sydney system. Of the 1006 patients examined, 34.5% had duodenal ulcer disease, 3.5% gastric ulcer disease, and 2% with coexistent ulceration. Most patients (50.2%) were classified as having non-ulcer dyspepsia. Altogether 2.4% of patients had gastric cancer and two further patients had carcinoma in the gastric stump. Of the ulcer disease patients, 87.2% had histological evidence of H pylori infection. After patients who had taken antibiotics or bismuth compounds in the preceding four weeks were excluded, 98.9% of the duodenal ulcer disease, 100% of the gastric ulcer disease, and 100% of the coexistent ulcer disease patients had evidence of H pylori infection. In patients with gastric cancer who had not taken antimicrobial agents in the four weeks before endoscopy, 83.3% had evidence of H pylori infection. Thus, there was a high rate of duodenal ulcer disease and a low rate of gastric ulcer disease in southern China, an area of low gastric cancer mortality. There was a specific topographical relationship between H pylori, the histological response, and gastroduodenal disease. Our data suggest that the status of a nation as either 'developed' or 'developing' can not be used to predict the upper gastrointestinal disease profile of its population.

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle.

Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.