Defective development and microcirculation of intestine in Npr2 mutant mice.

Intractable gastrointestinal (GI) diseases often develop during infancy. Our group previously reported that natriuretic peptide receptor B (NPR-B)-deficient Npr2slw/slw mice exhibit severe intestinal dysfunction, such as stenosis and distention, which resembles the dysfunction observed in Hirschsprung's disease-allied disorders. However, the root cause of intestinal dysfunction and the detailed of pathophysiological condition in the intestine are not yet clear. Here, we report that the intestine of preweaning Npr2slw/slw mice showed bloodless blood vessels, and nodes were found in the lymphatic vessel. Additionally, the lacteals, smooth muscle, blood vessel, and nerves were barely observed in the villi of preweaning Npr2slw/slw mice. Moreover, intramuscular interstitial cells of Cajal (ICC-IM) were clearly reduced. In contrast, villi and ICC-IM were developed normally in surviving adult Npr2slw/slw mice. However, adult Npr2slw/slw mice exhibited partially hypoplastic blood vessels and an atrophied enteric nervous. Furthermore, adult Npr2slw/slw mice showed markedly reduced white adipose tissue. These findings suggest that the cause of GI dysfunction in preweaning Npr2slw/slw mice is attributed to defective intestinal development with microcirculation disorder. Thus, it is suggested that NPR-B signaling is involved in intestinal development and control of microcirculation and fat metabolism. This report provides new insights into intractable GI diseases, obesity, and NPR-B signaling.

Accelerating effects of blebbistatin on relaxation process of cell membrane permeabilized trachea and taenia cecum from guinea pig.

Blebbistatin, a potent inhibitor of myosin II, is known to suppress smooth muscle contraction without affecting myosin light chain phosphorylation level. In order to clarify the regulatory mechanisms of blebbistatin on phasic and tonic smooth muscles in detail, we examined the effects of blebbistatin on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) trachea and taenia cecum preparations from guinea pigs. Blebbistatin significantly suppressed the force during relaxation both in skinned trachea and taenia cecum. The data fitting analysis of the relaxation processes indicates that blebbistatin accelerates slow (latch-like) bridge dissociation.

Changes in the Width of the Tibiofibular Syndesmosis Related to Lower Extremity Joint Dynamics and Neuromuscular Coordination on Drop Landing During the Menstrual Cycle.

BACKGROUND: Many injuries of the lower extremities, especially the knee and ankle, occur during sports activity, and the incidence rate is higher in women than in men. HYPOTHESIS: The hypothesis was that phases of the menstrual cycle affect the width of the tibiofibular syndesmosis during drop landing in healthy young women and that such changes at the tibiofibular joint also affect the dynamics and neuromuscular coordination of the lower extremities. STUDY DESIGN: Descriptive laboratory study. METHODS: Participants included 28 healthy young women (mean age, 21.0 ± 0.8 years). Blood samples were collected to determine plasma levels of estradiol and progesterone immediately before the performance of the task: drop landing on a single leg from a 30-cm platform. Using ultrasonography, the distance between the tibia and the distal end of the fibula, regarded as the width of the tibiofibular syndesmosis, was measured in an upright position without flexion of the ankle. The peak ground-reaction force (GRF) on landing was measured using a force platform. The time to peak GRF (Tp-GRF) was measured as the time from initial ground contact to the peak GRF. Hip, knee, and ankle joint angles during the single-leg landing were calculated using a 3-dimensional motion analysis system. Muscle activities of the lower extremities were measured using surface electromyography. RESULTS: The width of the tibiofibular syndesmosis was significantly greater in the luteal phase when compared with the menstrual, follicular, and ovulation phases (by 5%-8% of control). Also, during the luteal phase, the Tp-GRF was significantly shorter than in the follicular phase (by 6%); hip internal rotation and knee valgus were significantly greater than in the menstrual phase (by 43% and 34%, respectively); knee flexion was significantly less than in the menstrual and follicular phases (by 7%-9%); ankle dorsiflection was significantly less than in the follicular phase (by 11%); ankle adduction and eversion were significantly greater than in the menstrual and follicular phases (by 26%-46%, and 27%-33%, respectively); and activation of the gluteus maximus before landing was significantly lower than in the menstrual and follicular phases (by 20%-22%). CONCLUSION: The luteal phase appears to be associated with decreased strength and laxity of the ankle as well as lower extremity muscle activity in women. The changes presumably represent a greater risk for sports injuries. CLINICAL RELEVANCE: The results of this study suggest that the luteal phase may be related to the greater incidence of lower extremity injuries in women.

Instantaneous changes in respiratory function induced by passive pelvic suspension in the supine position in relation to increased diaphragm excursion.

[Purpose] This study aimed to introduce an approach of pelvic suspension (PS) using sling cords and to obtain evidence for changes in respiratory function of healthy subjects. [Subjects and Methods] Subjects were 25 healthy men. In the supine position, with hip and knee joints flexed at 90°, the subjects' pelvises were suspended with sling belts. Diaphragm excursion, respiratory function, and respiratory comfort in these postures were measured using ultrasonography, respirometry, and visual analog scale (VAS), respectively. [Results] When the pelvis was passively suspended with sling cords, the diaphragm moved 5 mm cranially and diaphragm excursion showed an instantaneous increase compared with the control. The tidal volume (VT) showed an increase and the respiration rate (RR) showed a decrease. The extent of diaphragm excursion was correlated with changes in VT under the control and PS conditions. Independent measurements of pulmonary function revealed that PS reduced the expiratory reserve volume, being correlated positively and negatively to increases in vital and inspiratory capacities, respectively. Furthermore, VAS values for respiratory ease were greater with PS than with the control. [Conclusion] These results suggest that PS effectively changed diaphragm excursion and respiratory function, leading to ease of breathing (i.e., deep and slow respiration).

Gender difference in distance of tibiofibular syndesmosis to joint dynamics of lower extremities during squat.

The incidence of lower extremity injury is greater in women than men, indicating gender difference in lower extremity function. Here we investigate the role of the ankle during squatting in young men and women by measuring the tibiofibular syndesmosis (TFS) distance and the angles of hip, ankle and knee joints. TFS distance was positively correlated to body mass in men, but not in women, suggesting some factor(s) affecting TSF distance in women. When divided into two groups with wide and narrow TFS distances, men apparently used ankle and hip joints evenly during squatting, but women, specifically those with a narrow TFS distance, used the hip joint more effectively than ankle. Estimated knee moment was positively correlated to TFS distance in women, but not in men. These results suggest that the women possessing a wider TFS distance use ankle function rather than hip junction when performing lower-extremity exercises (i.e. squatting), presumably accounting for the higher incidence of ankle injury in women during such activities.

Roles of basic amino acid residues in the activity of µ-conotoxin GIIIA and GIIIB, peptide blockers of muscle sodium channels.

To study in detail the roles of basic amino acid residues in the activity of µ-conotoxin GIIIA (µ-GIIIA) and GIIIB (µ-GIIIB), specific blockers of muscle sodium channels, seven analogs of µ-GIIIA, and two analogs of µ-GIIIB were synthesized. µ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg1, Arg13, and Arg19) with one, two, and three Lys residues. µ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys9, Lys11, Lys16, and Lys19) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg13 of µ-GIIIA was important for the activity, whereas that of Arg19 had little role for biological activity. Although [Arg9,11,16,19]µ-GIIIB showed higher activity than native µ-GIIIB, highly basic [Ala2,12, Arg9,11,16,19]µ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.

Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery.

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca(2+)] (27). This Ca(2+)-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca(2+) sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used alpha-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca(2+)-desensitizing hypoxic relaxation.

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice.

We previously showed that plasma membrane Ca(2+)-ATPase (PMCA) activity accounted for 25-30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca(2+) ([Ca(2+)](i)) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1(+/-), Pmca4(+/-), Pmca4(-/-), and Pmca1(+/-)Pmca4(-/-) mice. There were no differences in basal [Ca(2+)](i) values between bladder preparations. KCl (80 mM) elicited both larger forces (150-190%) and increases in [Ca(2+)](i) (130-180%) in smooth muscle from Pmca1(+/-) and Pmca1(+/-)Pmca4(-/-) bladders than those in WT or Pmca4(-/-). The responses to carbachol (CCh: 10 muM) were also greater in Pmca1(+/-) (120-150%) than in WT bladders. In contrast, the responses in Pmca4(-/-) and Pmca1(+/-)Pmca4(-/-) bladders to CCh were significantly smaller (40-50%) than WT. The rise in half-times of force and [Ca(2+)](i) increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4(-/-) (130-190%) and Pmca1(+/-)Pmca4(-/-) (120-250%) bladders, but not in Pmca1(+/-) bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca(2+) clearance, while PMCA4 is essential for the [Ca(2+)](i) increase and contractile response to the CCh receptor-mediated signal transduction pathway.

Ca2+-desensitizing hypoxic vasorelaxation: pivotal role for the myosin binding subunit of myosin phosphatase (MYPT1) in porcine coronary artery.

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We showed that at high stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in [Ca(2+)](i). This 'Ca(2+)-desensitizing hypoxic relaxation' was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)]. Rho kinase-dependent phosphorylation of MYPT1 (p-MYPT1) is associated with decreased MRLC phosphatase (MLCP) activity, and increased Ca(2+) sensitivity of both p-MRLC and force. We tested the hypothesis that hypoxia induces Ca(2+)-desensitizing hypoxic relaxation via dephosphorylation of p-MYPT1, consequently increasing MLCP activity and thus decreasing p-MRLC. alpha-Toxin-permeabilized PCASM pretreated with ATPgammaS did not relax in response to hypoxia. Moreover, when MRLC but not MYPT1 was protected from ATPgammaS thiophosphorylation by the MRLC kinase inhibitor ML7 (300 mum), hypoxia remained ineffective. In contrast, hypoxic relaxation was preserved with further addition of the Rho kinase inhibitor Y27632 (1 mum), to attenuate thiophosphorylation of MYPT1. Importantly, measurements of p-MRLC, and p-MYPT1 at T696 and T853 (human sequence) paralleled that of force. We conclude that Ca(2+)-desensitizing hypoxic relaxation requires dephosphorylation of p-MYPT1. Moreover, no kinases, other then those inhibited by ML7 and Y27632, nor their associated phosphoproteins can be involved in Ca(2+)-desensitizing hypoxic relaxation.

Role of plasma membrane Ca2+-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice.

We investigated the roles and relationships of plasma membrane Ca(2+)-ATPase (PMCA), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2, and Na(+)/Ca(2+) exchanger (NCX) in bladder smooth muscle contractility in Pmca-ablated mice: Pmca4-null mutant (Pmca4(-/-)) and heterozygous Pmca1 and homozygous Pmca4 double gene-targeted (Pmca1(+/-)Pmca4(-/-)) mice. Gene manipulation did not alter the amounts of PMCA1, SERCA2, and NCX. To study the role of each Ca(2+) transport system, contraction of circular ring preparations was elicited with KCl (80 mM) plus atropine, and then the muscle was relaxed with Ca(2+)-free physiological salt solution containing EGTA. We measured the contributions of Ca(2+) clearance components by inhibiting SERCA2 (with 10 microM cyclopiazonic acid) and/or NCX (by replacing NaCl with N-methyl-D-glucamine/HCl plus 10 microM KB-R7943). Contraction half-time (time to 50% of maximum tension) was prolonged in the gene-targeted muscles but marginally shortened when SERCA2 or NCX was inhibited. The inhibition of NCX significantly inhibited this prolongation, suggesting that NCX activity might be augmented to compensate for PMCA4 function in the gene-targeted muscles under nonstimulated conditions. Inhibition of SERCA2 and NCX as well as gene targeting all prolonged the relaxation half-time. The contribution of PMCA to relaxation was calculated to be approximately 25-30%, with that of SERCA2 being 20% and that of NCX being 70%. PMCA and SERCA2 appeared to function additively, but the function of NCX might overlap with those of other components. In summary, gene manipulation of PMCA indicates that PMCA, in addition to SERCA2 and NCX, plays a significant role in both excitation-contraction coupling and the Ca(2+) extrusion-relaxation relationship, i.e., Ca(2+) homeostasis, of bladder smooth muscle.

A phenoxazine compound, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one reverses the phenylephrine or high-K+ induced contraction of smooth muscles in rat aorta and guinea pig tenia cecum.

2-Amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1) reversed concentration dependently the contraction of both rat aorta and guinea pig tenia cecum induced by phenylephrine or high-K(+). Since Phx-1 suppresses the responses of human mononuclear cells to phytohemagglutinin, (Akazawa et al. Tohoku J. Exp. Med., 196, 185-192, 2002), Phx-1 may be useful for developing new vasorelaxing agents with immunosuppressive action.

Ca2+-independent hypoxic vasorelaxation in porcine coronary artery.

To demonstrate a Ca(2+)-independent component of hypoxic vasorelaxation and to investigate its mechanism, we utilized permeabilized porcine coronary arteries, in which [Ca(2+)] could be clamped. Arteries permeabilized with beta-escin developed maximum force in response to free Ca(2+) (6.6 microm), concomitant with a parallel increase in myosin regulatory light chain phosphorylation (MRLC-P(i)), from 0.183 +/- 0.023 to 0.353 +/- 0.019 MRLC-P(i) (total light chain)(-1). Hypoxia resulted in a significant decrease in both force (-31.9 +/- 4.1% prior developed force) and MRLC-P(i) (from 0.353 to 0.280 +/- 0.023), despite constant [Ca(2+)] buffered by EGTA (4 mm). Forces developed in response to Ca(2+) (6.6 microm), Ca(2+) (0.2 microm) + GTPgammaS (1 mM), or in the absence of Ca(2+) after treatment with ATPgammaS (1 mM), were of similar magnitude. Hypoxia also relaxed GTPgammaS contractures but importantly, arteries could not be relaxed after treatment with ATPgammaS. Permeabilization with Triton X-100 for 60 min also abolished hypoxic relaxation. The blocking of hypoxic relaxation after ATPgammaS suggests that this Ca(2+)-independent mechanism(s) may operate through alteration of MRLC-P(i) or of phosphorylation of the myosin binding subunit of myosin light chain phosphatase. Treatment with the Rho kinase inhibitor Y27632 (1 microm) relaxed GTPgammaS and Ca(2+) contractures; but the latter required a higher concentration (10 microm) for consistent relaxation. Relaxations to N(2) and/or Y27632 averaged 35% and were not additive or dependent on order. Our data suggest that the GTP-mediated, Rho kinase-coupled pathway merits further investigation as a potential site of this novel, Ca(2+)-independent O(2)-sensing mechanism. Importantly, these results unambiguously show that hypoxia-induced vasorelaxation can occur in permeabilized arteries where the Ca(2+) is clamped at a constant value.

Ca2+ clearance in smooth muscle: lessons from gene-altered mice.

The regulation of intracellular [Ca(2+)](i) is important for all cells, but in particular for smooth muscle, as [Ca(2+)](i) is a key second messenger leading to contraction. Mechanisms for the cellular clearance of [Ca(2+)](i) form one side of Ca(2+) homeostasis and include: Plasma Membrane Ca(2+) ATPases (PMCA), Sarcoplasmic/Endoplasmic Reticulum Ca(2+) ATPases (SERCA), Na(+)-Ca(2+)-exchangers (NCX) when coupled to the Na(+)-K(+) ATPases (NKA) and in some cases mitochondria. The nature and relative contribution of these various components of cytosolic Ca(2+) clearance have long been an important topic for study in smooth muscle, particularly as related to regulation of contractility. These studies have largely depended on inhibition of the various components. Recently advances in gene-targeting and transgenesis have made it possible to add or delete individual components, and importantly specific isoforms from the cell. In this brief review, we will focus on new information on Ca(2+) clearance in smooth muscle gained from studies on gene-altered mice models. These provide a deeper understanding of distinct functional roles for individual isoforms and the interactions between various components.

Presence of normal creatine in the muscle of a patient with a mutation in the creatine transporter: a case study.

To date, more than seven families have been reported who carry a mutation in the X-linked creatine-transporter (CrT) gene. The resulting lack of creatine in the brain is associated with mental retardation, severe expressive language disorder, mild epilepsy, and a complete absence of Cr in the brain (measured using MRS). Conversely, these patients had no observable cardiac or musculo-skeletal deficits. In this case study, a 22-year-old patient underwent surgical repair for scoliosis. Proton MRS of this patient's brain demonstrated the near-absence of creatine and phosphocreatine within the cerebral white and deep gray matter structures. Cerebral atrophy was noted with serial MRI examinations. Subsequent genetic and metabolic analysis showed some biochemical anomalies consistent with a CrT deficiency. The mutation in this patient was identified as a deletion at phenylalanine 107 (delF107). Control muscle biopsies were obtained from archived samples, which had been taken with informed consent during routine muscle biopsies for diagnostic purposes. We determined that the total Cr concentration in the skeletal muscle biopsy was 39.3 +/- 2.94 mmol/kg wet wt., which is not significantly different from non-CrT controls, n = 3 (43.3 +/- 3.57 mmol/kg wet wt.). We conclude that the brain appears to lack the ability to transport creatine when there is a mutation in the CrT gene. However, the muscle utilizes another mechanism for maintaining normal creatine levels. Identifying this alternative creatine-transport mechanism may be useful in treating the neurologic and cognitive impairments of patients with creatine-transporter deficiency.

Effects of metabolic inhibition on phosphorylation levels of PKC isoforms in the guinea pig taenia caeci.

We investigated which isoform of protein kinase C (PKC) is responsible to metabolic inhibition in the guinea pig taenia caeci with respect to their phosphorylation levels. By Western blot analysis using isoform-specific antibodies, at least four isoforms of PKC, alpha, beta2, epsilon and zeta were identified in the taenia. Prolonged metabolic inhibition of hypoxia, hypoxia+glucose depletion, and addition of cyanide (all in the presence of high K+) for more than 60 min, but not glucose-depletion only, elicited dephosphorylation of PKCs, alpha, beta2 and epsilon, except zeta. Ca2+ depletion from the medium prevented the dephosphorylation of PKCs induced by hypoxia, and apparently inhibited the dephosphorylation induced by hypoxia+glucose depletion. Acute treatment with hypoxia for 10-30 min elicited a gradual dephosphorylation of PKCbeta2, but not of other tested PKC isoforms. Considering the ATP level under various metabolic conditions reported previously, PKCbeta2 is suggested to be primarily responsible to hypoxia, and its dephosphorylation is closely associated with the alteration of adenylate compounds in the cell. Re-oxygenation after prolonged hypoxia did not restore the phosphoryation level of any tested PKCs, suggesting that the dephosphorylation of PKCs is associated with the irreversible damage of the cell under hypoxia. Presumably, the dephosphorylaton of PKCs, particularly PKCbeta2, plays a role in the signal transduction pathway under metabolic inhibition of the taenia, as reported in proliferative and pathophysiological processes in many other cells.

Hypoxic vasorelaxation: Ca2+-dependent and Ca2+-independent mechanisms.

The mechanisms of oxygen sensing in vascular smooth muscle have been studied extensively in a variety of tissue types and the results of these studies indicate that the mechanism of hypoxia-induced vasodilation probably involves several mechanisms that combined to assure the appropriate response. After a short discussion of the regulatory mechanisms for smooth muscle contractility, we present the evidence indicating that hypoxic vasorelaxation involves both Ca2+-dependent and Ca2+-independent mechanisms. More recent experiments using proteomic approaches in organ cultures of porcine coronary artery reveal important changes evoked by hypoxia in both Ca2+-dependent and Ca2+-independent pathways.

Down-regulation of an ankyrin repeat-containing protein, V-1, during skeletal muscle differentiation and its re-expression in the regenerative process of muscular dystrophy.

Using Western blot analysis and immunohistochemical methods, we examined the expression of V-1, a member of the ankyrin repeat-containing protein family, during differentiation and regeneration of skeletal muscle. The expression of V-1 was high in cultured myoblasts and decreased during their differentiation into myotubes, while high expression was maintained when muscle differentiation was inhibited by treatment with basic fibroblast growth factor. Down-regulation of V-1 also occurred during in vivo muscle differentiation from embryonic to postnatal stages, reaching an undetectable level in mature skeletal muscle. In contrast, strong V-1 immunoreactivity was detected again in myoblasts and regenerating muscle fibers with a small diameter, which were observed in Duchenne muscular dystrophy and its animal model, mdx mouse. Thus, it seems that V-1 is a good marker for early stage of muscle regeneration and changes of its expression suggest that V-1 plays a role in prenatal muscle differentiation and postnatal muscle regeneration.

V-1, a catecholamine biosynthesis regulatory protein, positively controls catecholamine secretion in PC12D cells.

Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K(+)-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca(2+)](i)) using fura-PE3, V-1 overexpression was observed to enhance high K(+)-elicited [Ca(2+)](i) elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K(+)-induced dopamine secretion by V-1 overexpression results from the potentiation of high K(+)-induced [Ca(2+)](i) elevation and the increase in the number of dense-cored vesicles.

A constitutively nuclear form of NFATx shows efficient transactivation activity and induces differentiation of CD4(+)CD8(+) T cells.

The Ca(2+) signal facilitates nuclear translocation of NFAT through the dephosphorylation of clustered serine residues in the calcium regulatory domain by the Ca(2+)/calmodulin-dependent phosphatase calcineurin. The conformation of dephosphorylated NFAT exposes the nuclear localization signal for translocation into the nucleus and masks the nuclear export sequence to keep the protein in the nucleus. It has been reported that deletion of some serine-rich motifs masking the nuclear localization signal results in the translocation of NFAT into the nucleus, but that the nuclear export sequence located at the N terminus also needs to be deleted for NFATx (NFAT4/NFATc3) to exert efficient transactivation function. Here, we report that deletion of the critical serine-rich motifs of NFATx leads to a conformation that efficiently exposes the nuclear localization signal and that has stronger transcription activity compared with the fully activated wild-type protein in the presence of the nuclear export sequence. This also suggests that the regulation of the transactivation domain by phosphorylation observed in NFAT1 may not contribute significantly to the transcription activity of NFATx. The expression of this constitutively nuclear form of NFATx in the CD4(+)CD8(+) T cell line facilitates differentiation into the CD4 single-positive stage upon stimulation with phorbol ester. Our data suggest that NFATx is involved in the regulation of co-receptor expression during differentiation into the CD4 single-positive stage.