Body composition at farrowing and nutrition during lactation affect the performance of primiparous sows: II. Milk composition, milk yield, and pig growth

We used 35 primiparous sows to investigate the link between body fatness at farrowing and voluntary feed intake (VFI) during lactation. Two groups of sows were fed differently throughout gestation (either 2.3 kg/d of a diet containing 5.8% CP and 14.6 MJ DE/kg as fed or 1.7 kg/d of a diet containing 15.6% CP and 14.5 MJ DE/kg as fed) so that they commenced lactation at a similar body weight (158 to 152 kg) but with different body compositions: either 340 (fat) or 280 (lean) g of body fat/kg BW (P < .001). During lactation, sows were offered either a low-protein diet (7.9% CP and 15.5 MJ DE/kg as fed) or a high-protein diet (19.0% CP and 15.6 MJ DE/kg as fed) on an ad libitum basis. During lactation, VFI was measured daily, and sow body weight and backfat were measured weekly. Blood samples were collected from sows on d 110 of gestation and d 14 and 28 of lactation, and plasma was analyzed for NEFA, glycerol, insulin, glucose, and beta-hydroxybutyrate. Fat sows ate 30% less than their lean counterparts during lactation (P < .001), which corresponded to a 70% higher concentration of NEFA in plasma (P = .01) and a 30% higher concentration of glycerol (P = .15). The VFI during the first 2 wk of lactation was affected only by body fatness and not by the protein content of the lactation diet. The dietary supply of protein influenced VFI during wk 3 and 4 of lactation, possibly by affecting milk production and hence the drive to consume feed. Weight loss, particularly lean tissue loss, was minimized by feeding the high-protein diet during lactation (P < .002).     

Alteration of milk fat by variation in the source and amount of starch in a total mixed diet fed to dairy cows

The effect on milk yield and composition of the supplementation of the diets of dairy cows with wheat or potato peelings was studied at three different starch intakes (< 5, 6, and > 7.5 kg/d) for dry matter intakes around 20 kg/d. Starch supply was varied using different dietary concentrations of corn silage. Trials were conducted using Latin square designs, and all cows were fed a total mixed diet composed of corn silage, grass silage, or both; soybean meal; a mixed meal with formaldehyde treatment; minerals; and one of the following energy concentrates: potato peelings or wheat. Dry matter and energy intakes varied significantly only at the low starch concentration; higher intakes were observed when the potato peelings were fed. Body weight, milk yield, true protein content, lactose content, and protein yield were not affected by treatment. Milk fat content was higher (+3.3 g/kg) when potato peelings were fed at the high starch concentration but was unaffected at the low and medium starch concentration. Slow degradation of starch from the potato peelings in the rumen could enhance a higher delivery of precursors of milk fat synthesis in the udder. The effect on fat yield differed among starch concentrations. Milk fat content appeared to decrease for cows fed diets containing quickly degradable starch at a starch intake > 7 kg/d.     

S-adenosylmethionine decarboxylase activity and utilization of exogenous putrescine are enhanced in colon cancer cells stimulated to grow by EGF

Polyamines spermidine and spermine and their precursor putrescine are necessary for cell growth. Polyamine content is high in rapidly growing malignant cells, due to enhanced putrescine synthesis by ornithine decarboxylase (ODC), and increased uptake. In contrast to other cells of the body, colon cancer cells are exposed to high putrescine concentrations from the lumen. AIMS: To investigate the utilization of luminal putrescine in colon cancer, we studied the effect of a potent mitogen, epidermal growth factor (EGF), on the activity of the enzyme responsible for putrescine conversion, S-adenosylmethionine decarboxylase (SAMDC), in Caco-2 cells. METHODS: Cell counts, ODC and SAMDC activities and intracellular polyamines were evaluated in the presence and absence of exogenous putrescine in concentrations resembling those normally present in the colonic lumen. RESULTS: ODC and SAMDC activity and putrescine uptake were strongly stimulated by EGF. Both synthesized and absorbed putrescine was rapidly converted to spermidine and spermine after EGF. Conversion pattern was identical in the cells stimulated with EGF only and EGF plus exogenous putrescine, indicating that, if stimulated to proliferate, colon cancer cells utilize the entire available putrescine pool. SAMDC inhibitor, methylglyoxal-bis-guanylhydrazone, induced growth arrest which was not reversed by exogenous putrescine, but only by high concentrations of spermidine. CONCLUSION: Enhanced proliferation in colon cancer cells is associated with increased SAMDC activity and rapid conversion of putrescine to spermidine and spermine. SAMDC might be a preferable target for therapeutic attempts to impair growth by reducing intracellular polyamine pools in colon cancer.     

The effect of enhanced milk yield of dairy cows by frequent milking or suckling on intake and digestibility of the diet

The prevalence of osteoporosis and the incidence of fractures are substantially lower in black than in white subjects, a finding generally attributed to racial differences in adult bone mass. Whether these racial differences are present in childhood is the subject of considerable interest, as the amount of bone gained during growth is a major determinant of future susceptibility to fractures. We measured the density and size of the vertebrae and femurs of 80 black and 80 white healthy children, 8-18 yr of age, matched for age, gender, height, weight, and stage of sexual development, using computed tomography. Race had a significant and differential effect on the bones in the axial and appendicular skeletons. In the axial skeleton, black children had greater cancellous bone density, but similar cross-sectional area of the vertebral bodies. In contrast, in the appendicular skeleton, black children had greater femoral cross-sectional area, but similar cortical bone area and cortical bone density. Compared to white children, vertebral bone density and femoral cross-sectional area at sexual maturity were, on the average, 10.75% and 5.7% higher, respectively, in black children. Such significant variations may contribute to the racial differences in the prevalence of osteoporosis between black and white adults.     

The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity

Osborne-Mendel (OM) and S5B/Pl rats differ in their sensitivity to develop obesity when fed a high fat (HF) diet; OM rats become obese, whereas S5B/Pl rats remain thin. We have investigated the possibilities that either an impaired leptin response or resistance to leptin action underlies the sensitivity to this form of obesity in OM rats. In Experiment 1, OM and S5B/Pl rats fed a nonpurified diet were killed at d 0 or were fed either a HF (56% fat energy) or a low fat (LF, 10% fat energy) diet for 2 or 7 d. The HF diet increased serum leptin significantly by d 2 to levels that were similar in both rat strains. At 7 d, leptin levels were lower than at d 2 but remained higher than levels in the d 0 control groups. The leptin mRNA:18S RNA ratio in epididymal adipose tissue increased to higher levels in HF-fed OM rats than in S5B/Pl rats fed that diet. However, although the LF diet had no effect in S5B/Pl rats, it increased leptin mRNA levels in epididymal adipose tissue of OM rats compared with the controls fed the nonpurified diet. In Experiment 2, OM and S5B/Pl rats were fed HF or LF diets for 5 wk. At that time, their feeding response to a range of leptin doses (0, 1, 5 or 10 microgram) given intracerebroventricularly was tested after overnight food deprivation. There was a similar dose-dependent reduction in energy intake in response to leptin in both OM and S5B/Pl rats. These responses were independent of the diet. The data suggest that the susceptibility of OM rats to HF diet-induced obesity is not related to either a loss of central sensitivity to leptin or a failure to enhance leptin production acutely, although the failure to maintain chronically increased levels of serum leptin could contribute to the obesity.     

Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Unrecognized and untreated depression occurs frequently in Alzheimer's disease (AD) patients, adding to the agony already experienced by patient and caregiver. The authors screened AD patients living with a family caregiver for depression. Twelve patients with confirmed depression were treated in an open-label study with antidepressant medication, with dosage adjustment by a psychiatrist at Weeks 2, 4, 8, and 16. Data collection occurred at baseline, Week 4, and Week 16. Depression decreased significantly (p < .01). Contrary to expectations, patient functional capacity declined (p = .045). Cognition remained unchanged (p > .05). Caregiver burden, caregiver depressive symptomatology, and quality of life of patient and caregiver remained unchanged (p > .05). The authors conclude that depression in AD can be detected if a collateral source, such as the caregiver, is available. The depression can and should be treated. More research is needed to determine the impact on patient functioning, caregiver burden, caregiver depressive symptomatology, and quality of life of patient and caregiver.     

Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane

A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.     

Plasma concentrations of risperidone and its 9-hydroxy metabolite and their relationship to dose in schizophrenic patients: simultaneous determination by a high performance liquid chromatography with electrochemical detection

A simple, sensitive and accurate method for the simultaneous determination of risperidone (RSP) and its 9-hydroxy metabolite (9-OH-RSP) in human plasma is described. The relationship between dose of RSP and the plasma concentration of RSP and 9-OH-RSP in a clinical situation is discussed. Both compounds were isolated from plasma by a simple one-step liquid-liquid extraction with 15% methylene chloride in pentane. High-performance liquid chromatography separations were made on a cyano column and the compounds were detected by electrochemical detector. The method had sufficient sensitivity to determine RSP and 9-OH-RSP accurately at concentrations as low as 0.25 ng/ml when 1 ml of plasma is used for the analysis. The assay determinations were accurate, precise and consistent with a coefficient of variation less than 15%. Commonly co-administered drugs and other antipsychotics did not interfere with the analysis of either RSP or 9-OH-RSP There were large variations in inter- and intra-individual values of plasma concentrations of RSP and 9-OH-RSP. The 9-OH-RSP appears to be the major circulating active moiety and its plasma concentrations were, on the average 22 fold higher than that of RSP in schizophrenic patients treated with RSP. The ratio of RSP/9-OH-RSP concentrations suggested that three of the patients may have deficiency in cytochrome P450 enzyme CYP 2D6. The plasma concentrations of RSP showed a weak relationship with the administered daily oral dose (r = 0.4684, p = 0.01, n = 215). However, there was a good relationship between the daily dose of RSP and the plasma concentration of 9-OH-RSP (r = 0.6654, p = 0.01, n = 280) or the total active moiety, sum of RSP and 9-OH-RSP concentrations (r = 0.7041, p = 0.0005, n = 280). The measurement of the total active moiety in plasma of schizophrenic patients may be useful for assessing the relationship between dose and plasma concentration and dose and clinical outcome of patients rather than measuring RSP alone.     

Effects of resistance to milk flow and the provision of hay on nonnutritive sucking by dairy calves

This study examined effects of resistance to milk flow and the provision of hay on the duration of nutritive sucking and subsequent nonnutritive sucking by dairy calves. In a series of four experiments, 12 male Holstein calves were individually fed milk from an artificial teat. Resistance to milk flow was varied by adjusting the orifice size within the milk supply tube. Using a Latin square design, each calf was fed the same quantity of milk using four orifice sizes (one per day for 4 consecutive d). The duration of nutritive sucking (time required to finish the milk meal) was longer when calves were fed from the smallest orifice size (0.16-cm diameter) than when calves were fed from the largest orifice size (0.55-cm diameter). Calves compensated for resistance to milk flow in an attempt to maintain milk intake; however, changes in sucking rate alone were probably not responsible for observed differences in the rate of milk consumption from different orifice sizes. The duration of nonnutritive sucking (sucking of the teat following the milk meal) was significantly reduced when calves were fed from the smallest orifice compared with that when calves were fed from the largest orifice. Nonnutritive sucking was reduced but not eliminated even with the longest meal duration. The provision of hay to calves following the meal also significantly reduced the amount of nonnutritive sucking. Reduction in flow rate when calves drink milk through a teat and the provision of hay after the meal can reduce the incidence of nonnutritive sucking following the meal and may help to limit cross-sucking in group housing systems.     

Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-trans-retinoic acid

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-trans-retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 micrograms of atRA per g of diet (230 micrograms per rat per day) or 250 micrograms of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 micrograms of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 micrograms of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 micrograms/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 micrograms of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 micrograms of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 micrograms of atRA per g of diet fed to VAD dams (approximately 4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.     

The release of a coronary vasodilator metabolite from the guinea-pig isolated perfused heart stimulated by catecholamines, histamine and electrical pacing and by exposure to anoxia

The results of surveys and inquiries to identify autistic children, carried out in England and Wales, the U.S.A. and Denmark, are compared. Three studies, in each of which either a total population of children or a wide range of handicapped children was screened, using case-note inspection and interviews, all estimated the prevalence of the autistic syndrome to be between four and five children per 10,000 aged under 15 years. Inquiries that counted diagnosed cases only or that relied upon local authority records produced much lower prevalence rates for the autistic syndrome. The reasons for this are examined, and the implications for prevalence studies of handicapping conditions are discussed.     

Cell death and cell proliferation contribute to the enhanced growth of foci by fasting in rat medial colon

We have recently shown that fasting before initiation markedly stimulated the growth of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the rat medial colon. Here we investigated the mechanisms by which fasting enhanced the growth of ACF. Rats were exposed to 4 day-starvation, then they were given AOM (20 mg/kg) on the first day of refeeding. 4 day-fasting depressed cell proliferation as shown by the decreased mitotic index and enhanced cell death by apoptosis. On the first day of refeeding, apoptotic index remained higher than control values, while mitotic index markedly increased in the colonic epithelium of fasted/ refed rats. The administration of AOM induced an apoptotic wave, that was higher in controls, and a transient drop in the mitotic index that recovered quickly in the fasted/refed group. These data suggest that starvation-induced apoptosis represents the mitogenic stimulus to increase the rates of cell proliferation responsible for the enhanced growth of ACF in fasted/refed rats.     

Interactions between dietary fat type and xylanase supplementation when rye-based diets are fed to broiler chickens 2. Performance, nutrient digestibility and the fat-soluble vitamin status of livers

We analyzed retrospectively the outcome of 169 patients in chronic hemodialysis (CHD), divided into four groups: 1) 24 patients with diabetic nephropathy (age 53.7 +/- 11 years); 2) 19 with polycystic kidney disease (age 55.3 +/- 9 years) 3) 43 patients older than 60 when starting chronic hemodialysis with etiologies different from diabetes and polycystic kidney disease (age 69.2 +/- 5.8 years) and 4) 83 patients younger than 60 with diverse etiologies (age 42.8 +/- 12.4 years). In groups 1, 2 and 3 serum creatinine, arterial hypertension at the beginning, morbility, mortality and its causes were registered. In group 1, the prevalence of severe diabetic retinopathy and cardiovascular disease at the beginning were also analyzed. In all groups survival was determined. Of the diabetics, 92% presented severe diabetic retinopathy and 88% cardiovascular disease. The prevalence of hypertension was 100, 74 and 67% in groups 1, 2 and 3, respectively (p = 0.13). Twelve diabetics died before the first year of treatment; there was no difference in creatinine, age, cardiovascular disease, severe retinopathy and hypertension with those who lived more than one year. The percentage of time in risk hospitalized and the days/patients/year hospitalized were significantly different between group 1 and 3 and group 2 (p < 0.001). Patients were hospitalized for similar causes in groups 1 and 3: the initiation of CHD, cardiovascular and neurological diseases. The main causes of death in groups 1 and 3 were: cardiovascular disease and sudden death at home. Survival was better in group 2 compared with group 1 (p = 0.0014) but was similar between groups 1 and 3 (p = 0.21) even though there was a difference of 15 years between them. The Cox's proportional hazard model identified as risk factors diabetes, age, year of starting chronic hemodialysis and hospitalization episodes, adjusted for covariates. The outcome of diabetic patients in chronic hemodialysis showed high morbidity and mortality and was quite similar to that of elderly patients.     

Measurement of serum isopropanol and the acetone metabolite by proton nuclear magnetic resonance: application to pharmacokinetic evaluation in a simulated overdose model

The regional distribution of [11C]d-threo-methylphenidate in mouse brain was very similar to that of [3H]WIN 35,428 ((-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane), and the two radioligands were displaced from striatum similarly after administration of the potent cocaine analog RTI-55 ((-)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane). However, while striatal [3H]WIN 35,428 increased between 5 and 30 min, striatal [11C]d-threo-methylphenidate halved. Thus [11C]d-threo-methylphenidate binds similarly to but more reversibly than [3H]WIN 35,428. The methyl ester of L-DOPA (L-3,4-dihydroxyphenylalanine; 200 mg/kg) plus benserazide plus clorgyline, which markedly elevates rat striatal extracellular dopamine (Wachtel and Abercrombie, 1994, J. Neurochem. 63, 108), decreased the mouse striatum-to-cerebellum ratio for [11C]d-threo-methylphenidate at 30 min by 13% (P < 0.05). In positron emission tomographic (PET) baboon studies [11C]d-threo-methylphenidate binding was insensitive to drugs expected to lower endogenous dopamine. These experiments suggest that normal synaptic dopamine does not compete for binding with [11C]d-threo-methylphenidate, and will not affect PET measures of dopamine transporter availability.     

Full-spreading platelets induced by the recombinant rhodostomin are via binding to integrins and correlated with FAK phosphorylation

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.     

Binding of factor VIII to von willebrand factor is enabled by cleavage of the von Willebrand factor propeptide and enhanced by formation of disulfide-linked multimers

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).     

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.