Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.

Previously we showed that atrial natriuretic factor (ANF) decreases cardiac cell volume by inhibiting ion uptake by Na+/K+/2CI-cotransport. Digital video microscopy was used to study the role of guanosine 3',5'-monophosphate (cGMP) in this process in rabbit ventricular myocytes. Each cell served as its own control, and relative cell volumes (volumetest/volumeco,trol) were determined. Exposure to 10 IxM 8-bromo-cGMP (8-Br-cGMP) reversibly decreased cell volume to 0.892 -0.007; the EDs0 was 0.77 +-0.33 tzM. Activating guanylate cyclase with 100 IxM sodium nitroprusside also decreased cell volume to 0.889 ---0.009. In contrast, 8-bromo-adenosine 3',5'-monophosphate (8-Br-AMP; 0.01-100 tzM) neither altered cell volume directly nor modified the response to 8-Br-cGMP. The idea that cGMP decreases cell volume by inhibiting Na+/K+/2C1-cotransport was tested by blocking the cotransporter with 10 I~M bumetanide (BUM) and removing the transported ions. After BUM treatment, 10 IxM 8-Br-cGMP failed to decrease cell volume. Replacement of Na ÷ with N-methyl-D-glucamine or C1-with methanesulfonate also prevented 8-Br-cGMP from shrinking cells. The data suggest that 8-Br-cGMP, like ANF, decreases ventricular cell volume by inhibiting Na+/K+/2C1-cotransport. Evidence that ANF modulates cell volume via cGMP was also obtained. Pretreatment with 10 IzM 8-Br-cGMP prevented the effect of 1 IzM ANF on cell volume, and ANF suppressed 8-Br-cGMP-induced cell shrinkage. Inhibiting guanylate cyclase with the quinolinedione LY83583 (10 tzM) diminished ANF-induced cell shrinkage, and inhibiting cGMP-specific phosphodiesterase with M&B22948 (Zaprihast; 100 IzM) amplified the volume decrease caused by a low dose of ANF (0.01 I~M) approximately fivefold. In contrast, neither 100 I~M 8-Br-cAMP nor 50 tzM forskolin affected the response to ANF. The effects of ANF, LY83583, and M&B29948 on cGMP levels in isolated ventricular myocytes were confirmed by 125I-cGMP radioimmunoassay. These data argue that ANF shrinks cardiac cells by increasing intracellular cGMP, thereby inhibiting Na+/K+/2C1-cotransport. Basal cGMP levels also appear to modulate cell volume.

1. Salbutamol, a fl2-agonist, increased the weight of the canine latissimus dorsi muscle. It also increased fusion frequency, and decreased time-to-peak tension, half-relaxation time, and total contraction time. These changes in twitch times and fusion frequency were associated with changes in the levels of proteins expressed in slow-and fast-twitch fibres. Salbutamol decreased the levels of the slow-twitch cardiac isoform of sarco-/endoplasmic reticulum Ca2P-ATPase (SERCA2a) and phospholamban proteins, and increased the level of the fast-twitch isoform of sarco-/endoplasmic reticulum Ca2+-ATPase (SERCAla).2. Changes in the levels of SERCA proteins, particularly SERCAla, could account for most of the increases in calcium uptake rate observed in homogenates of muscles from the salbutamol-treated animals and could partially account for the changes in half-relaxation rates and other twitch times.3. Changes in the levels of SERCAla, SERCA2a and phospholamban protein did not always follow changes in the levels of their corresponding mRNAs. Divergence depended upon the SERCA isoform and muscle. The muscles studied were latissimus dorsi and vastus intermedius. 4. Salbutamol did not change the level of myosin heavy chain (HC)-I isoforms in either muscle, suggesting that it did not increase the proportion of slow-twitch fibres in these muscles. It did increase the level of HC-IIx and decrease the level of HC-IIa isoforms in the latissimus dorsi. Salbutamol did not produce these effects in the vastus intermedius. It is of particular interest that salbutamol changed the relative levels of SERCA proteins in the latissimus dorsi muscle without producing significant changes in the level of HC-I isoform.

Chronic stimulation of a predominantly fast skeletal muscle enhanced the expression of type I (slow muscle) Ca-ATPase and suppressed the expression of the type II (fast muscle) Ca-ATPase. Monoclonal antibodies IID and IIHI 1 against type I (slow) and type II (fast) isoxymes respectively, were used to type the Ca-ATPases of the isolated SR (sarcoplasmic reticulum) by Western blots, and the Ca-ATPases of the muscle fibers by immuno~st~h~s~y.Of the fibers from control muscles 8096 stained for the type II isoxyme and 20% for the type I isoxyme. Following chronic stimulation all fibers stained for type I isoxyme and none stained for type II isoxyme. Ca-ATPase isoxyme distribution in isolated SR confirmed this effect of chronic stimulation. The calcium uptake activities of homogenates of stimulated muscles were 22% of the control muscles. The Ca-ATPase and calcium-uptake activities of the isolated SR from stimulated muscles were, respectively, 32 and 45% of the control muscles.

The calcium flux through an aqueous compartment was determined using a flow-through dialysis cell in which two dialysis membranes isolated the aqueous compartment. Addition of the intestinal vitamin D-dependent calcium-binding protein (CaBP) significantly enhanced the calcium flux at near physiological calcium concentrations (1 X 10(-6) M). Bovine serum albumin had no effect on the calcium flux. CaBP appears to be one of a class of low molecular weight, soluble binding proteins that enhance ligand diffusion.