Inflammation dynamically regulates steroid hormone metabolism and action within macrophages in rheumatoid arthritis.

RATIONALE: In inflammatory diseases such as rheumatoid arthritis (RA), steroid metabolism is a central component mediating the actions of immuno-modulatory glucocorticoids and sex steroids. However, the regulation and function of cellular steroid metabolism within key leukocyte populations such as macrophages remain poorly defined. In this study, the inflammatory regulation of global steroid metabolism was assessed in RA macrophages. METHODS: Bulk RNA-seq data from RA synovial macrophages was used to assess transcripts encoding key enzymes in steroid metabolism and signalling. Changes in metabolism were assessed in synovial fluids, correlated to measures of disease activity and functionally validated in primary macrophage cultures. RESULTS: RNA-seq revealed a unique pattern of differentially expressed genes, including changes in genes encoding the enzymes 11ß-HSD1, SRD5A1, AKR1C2 and AKR1C3. These correlated with disease activity, favouring increased glucocorticoid and androgen levels. Synovial fluid 11ß-HSD1 activity correlated with local inflammatory mediators (TNFα, IL-6, IL-17), whilst 11ß-HSD1, SRD5A1 and AKR1C3 activity correlated with systemic measures of disease and patient pain (ESR, DAS28 ESR, global disease activity). Changes in enzyme activity were evident in inflammatory activated macrophages in vitro and revealed a novel androgen activating role for 11ß-HSD1. Together, increased glucocorticoids and androgens were able to suppress inflammation in macrophages and fibroblast-like-synoviocytes. CONCLUSIONS: This study underscores the significant increase in androgen and glucocorticoid activation within inflammatory polarized macrophages of the synovium, contributing to local suppression of inflammation. The diminished profile of inactive steroid precursors in postmenopausal women may contribute to disturbances in this process, leading to increased disease incidence and severity.

The hallmarks of osteoarthritis and the potential to develop personalised disease-modifying pharmacological therapeutics.

Osteoarthritis (OA) is an age-related condition and the leading cause of pain, disability and shortening of adult working life in the UK. The incidence of OA increases with age, with 25% of the over 50s population having OA of the knee. Despite promising preclinical data covering various molecule classes, there is regrettably at present no approved disease-modifying OA drugs (DMOADs). With the advent of next generation sequencing technologies, other therapeutic areas, in particular oncology, have experienced a paradigm shift towards defining disease by its molecular composition. This paradigm shift has enabled high resolution patient stratification and supported the emergence of personalised or precision medicines. In this review we evaluate the potential for the development of OA therapeutics to undergo a similar paradigm shift given that OA is increasingly being recognised as a heterogeneous disease affecting multiple joint tissues. We highlight the evidence for the role of these tissues in OA pathology as different "hallmarks" of OA biology and review the opportunities to identify and develop targeted disease-modifying pharmacological therapeutics. Finally, we consider whether it is feasible to expect the emergence of personalised disease-modifying medicines for patients with OA and how this might be achieved.

An in situ FTIR spectroscopic study of the electrochemical oxidation of ethanol at a Pb-modified polycrystalline Pt electrode immersed in aqueous KOH.

A study of the mechanism of ethanol oxidation in alkaline solution on a platinum electrode modified with an irreversibly-deposited layer of lead has been carried out using in situ FTIR spectroscopy. This study provides support for the suggestion that the adsorption mechanism of ethanol is substantially modified in the presence of Pb, with a carbon-bonded intermediate being favoured leading to facile scission of the C-C bond in ethanol. We have found that the formation of carbonate takes place at potentials close to the thermodynamic value. At higher potentials, when Pb is lost to solution, the mechanism of oxidation of ethanol reverts to that found on a normal polycrystalline Pt surface, with the primary product being acetate.

Hyperimmune egg yolk antibodies developed against Clostridium perfringens antigens protect against necrotic enteritis.

Necrotic enteritis (NE) is a widespread infectious disease caused by Clostridium perfringens that inflicts major economic losses on the global poultry industry. Due to regulations on antibiotic use in poultry production, there is an urgent need for alternative strategies to mitigate the negative effects of NE. This paper presents a passive immunization technology that utilizes hyperimmune egg yolk immunoglobulin Y (IgY) specific to the major immunodominant antigens of C. perfringens. Egg yolk IgYs were generated by immunizing hens with 4 different recombinant C. perfringens antigens, and their protective effects against NE were evaluated in commercial broilers. Six different spray-dried egg powders were produced using recombinant C. perfringens antigens: α-toxin, NE B-like toxin (NetB; EB), elongation factor-Tu (ET), pyruvate:ferredoxin oxidoreductase, a mixture of 4 antigens (EM-1), and a nonimmunized control (EC). The challenged groups were either provided with different egg powders at a 1% level or no egg powders (EN). The NE challenge model based on Eimeria maxima and C. perfringens dual infection was used. In Experiments 1 and 2, the EB and ET groups exhibited increased body weight gain (BWG; P < 0.01), decreased NE lesion scores (P < 0.001), and reduced serum NetB levels (P < 0.01) compared to the EN and EC groups. IgY against NetB significantly reduced Leghorn male hepatocellular cytotoxicity in an in vitro test (P < 0.01). In Experiment 3, the protective effect of the IgYs mixture (EM-2) against C. perfringens antigens (NetB and EFTu) and Eimeria antigens (elongation factor-1-alpha: EF1α and Eimeria profilin: 3-1E) was tested. The EM-2 group showed similar body weight, BWG, and feed intake from d 7 to 22 compared to the NC group (P < 0.05). On d 20, the EM-2 group showed comparable intestinal permeability, NE lesion scores, and jejunal NetB and collagen adhesion protein levels to the NC group (P < 0.05). In conclusion, dietary mixture containing antibodies to NetB and EFTu provides protection against experimental NE in chickens through passive immunization.

Beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle does not modulate disease severity in the rodent meniscectomy model of osteoarthritis.

OBJECTIVE: To examine whether beta2-adrenergic agonist-induced hypertrophy of the quadriceps skeletal muscle can modulate the severity of osteoarthritis (OA) in the rodent meniscectomy (MNX) model. METHODS: Male Lewis rats were subcutaneously administered with 1.5 mg/kg/day clenbuterol hydrochloride (n=15) or saline vehicle (n=20) for 14 days. Following pre-treatment, five animals from each group were sacrificed to assess the immediate effects of clenbuterol. The remaining animals underwent either invasive knee surgery (clenbuterol pre-treated n=10; saline pre-treated n=10) or a sham control surgical procedure (saline pre-treated n=5). During disease initiation and progression, weight bearing was assessed by hindlimb loading. Myosin heavy chain (MHC) protein isoforms were quantified by silver stained SDS PAGE. OA severity was graded by assessment of toluidine blue stained step coronal sections of the total knee joint. RESULTS: Clenbuterol treatment resulted in an increase in total bodyweight, growth rate and in quadriceps skeletal muscle mass. Meniscal surgery resulted in the development of OA-like lesions, changes to weight bearing, and changes in MHC protein expression in the quadriceps. Clenbuterol-induced skeletal muscle hypertrophy had no effect on either weight bearing or articular pathology following MNX surgery. CONCLUSIONS: Our data reveal that clenbuterol-induced skeletal muscle hypertrophy is unable to mimic the beneficial clinical effects of increased musculature derived through targeted strength training in humans, in a rodent model of MNX-induced OA. In addition we observed fibre-type switching to "slow twitch" in the quadriceps muscle during the induction of OA that warrants further investigation as to its relationship to joint stability.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) modulates key biological pathways associated with OA disease pathology.

OBJECTIVE: To examine the role of mitogen-activated protein kinase-activated protein kinase 2 (MK2) in mediating the cellular response to pro-inflammatory cytokines in human primary osteoarthritis (OA) chondrocytes. METHODS: Delivery of a dominant negative MK2 was achieved in HeLa cells by adenoviral infection. Cellular heat shock protein (HSP27) activity was determined using a Bioplex assay. Primary OA chondrocytes were isolated by collagenase digestion of human articular cartilage. Phosphorylated MK2 was detected by immunoblotting and immunohistology. Transfection of primary chondrocytes with siRNA was achieved using cationic lipid and gene expression determined by real-time polymerase chain reaction. Production of prostaglandin E2 (PGE2) and matrixmetalloproteases (MMPs) was measured by enzyme-linked immunosorbent assay. RESULTS: Over-expression of a dominant negative MK2 inhibited HSP27 phosphorylation and significantly reduced both interleukin 1 (IL-1)beta and tumour necrosis factor (TNF)-alpha mediated release of PGE2 in HeLa cells over a 24h period. Phosphorylated MK2 was detected in OA articular cartilage and in isolated primary OA chondrocytes, where it was induced by IL-1beta. Transfection of OA chondrocytes with MK2 siRNA antisense significantly reduced both basal and IL-1beta induced PGE2 release. siRNA mediated MK2 knockdown also significantly reduced both basal and IL-1beta induced MMP13 expression and MMP13 and MMP3 protein release but had no effect on MMP1. CONCLUSIONS: Our data reveal that MK2 is active in OA human articular cartilage and in isolated primary human chondrocytes and that MK2 mediates the release of PGE2, MMP3 and MMP13. These findings suggest a role for MK2 in contributing to OA algesia and OA joint structural deterioration by mediating the downstream effects of p38 activation on PGE2 release and the expression and release of catabolic proteases.

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE: To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS: Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS: We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis.

BACKGROUND: Patients with rheumatoid arthritis (RA) experience extra-articular manifestations including osteoporosis and muscle wasting, which closely associate with severity of disease. Whilst therapeutic glucocorticoids (GCs) reduce inflammation in RA, their actions on muscle and bone metabolism in the context of chronic inflammation remain unclear. We utilised the TNF-tg model of chronic polyarthritis to ascertain the impact of therapeutic GCs on bone and muscle homeostasis in the context of systemic inflammation. METHODS: TNF-tg and wild-type (WT) animals received either vehicle or the GC corticosterone (100 µg/ml) in drinking water at onset of arthritis. Arthritis severity and clinical parameters were measured, serum collected for ELISA and muscle and bone biopsies collected for µCT, histology and mRNA analysis. In vivo findings were examined in primary cultures of osteoblasts, osteoclasts and myotubes. RESULTS: TNF-tg mice receiving GCs showed protection from inflammatory bone loss, characterised by a reduction in serum markers of bone resorption, osteoclast numbers and osteoclast activity. In contrast, muscle wasting was markedly increased in WT and TNF-tg animals receiving GCs, independently of inflammation. This was characterised by a reduction in muscle weight and fibre size, and an induction in anti-anabolic and catabolic signalling. CONCLUSIONS: This study demonstrates that when given in early onset chronic polyarthritis, oral GCs partially protect against inflammatory bone loss, but induce marked muscle wasting. These results suggest that in patients with inflammatory arthritis receiving GCs, the development of interventions to manage deleterious side effects in muscle should be prioritised.

On the resting potential of isolated frog sympathetic neurons.

One of the oldest questions of electrophysiology, the origin of the resting potential, has yet to be answered satisfactorily for most cells. Isolated frog sympathetic neurons, studied with whole-cell recording, generally have resting potentials of approximately -75 mV with an input resistance of approximately 300 M omega. These properties are not expected from the M-type K+ current (IM) or from other ionic currents previously described in these cells. In the -60 to -110 M mV voltage region, at least three currents are present: an inwardly rectifying current (IQ), a resting current with little voltage sensitivity carried at least in part by K+, and a (Na+,K+)ATPase pump current. The resting K+ current, not IM or IQ is the primary ionic current near the resting potential under these conditions. The electrogenic pump contributes an additional approximately 10 mV of hyperpolarization.

LHRH and GTP-gamma-S modify calcium current activation in bullfrog sympathetic neurons.

LHRH (chicken II luteinizing hormone-releasing hormone) partially reduces calcium currents and slows the activation kinetics of part of the remaining current in frog sympathetic neurons. The effects of LHRH are mimicked by intracellular dialysis with GTP-gamma-S. A strong depolarization can temporarily reverse the effects of LHRH or GTP-gamma-S: activation kinetics return to normal, and the amplitude of the current is increased (facilitation). Facilitation develops rapidly (tau = 4-6 ms at greater than +30 mV) and decays more slowly (t 1/2 = 60 ms at -80 mV). Tail currents in LHRH are smaller and faster than in the control, and these effects are partially reversed by facilitation. These results can be explained by a model in which a fraction of the channels is shifted into a "reluctant" gating mode, where opening requires stronger depolarization. If this mechanism is at the root of presynaptic inhibition, our results predict that inhibition of transmitter release would be overcome during bursts of high frequency activity.

Reevaluation of Ca2+ channel types and their modulation in bullfrog sympathetic neurons.

With 90 mM Ba2+, the main Ca2+ current in frog sympathetic neurons peaks near +30 mV and is blocked by omega-conotoxin GVIA (omega-CgTx). It is modulated by norepinephrine (NE) in a voltage-dependent manner via a membrane-delimited mechanism. Surprisingly, a different current dominates at more negative voltages (-30 to +10 mV). That novel current is not sensitive to selective blockers of L- or N-type channels (respectively, dihydropyridines or omega-CgTx) and is inhibited weakly if at all by NE. It is selectively inactivated at -40 mV and is selectively blocked by Ni2+, whereas Cd2+ is slightly more potent against the main current. The novel current is associated with a 19 pS channel (0.6 pA at 0 mV). This channel may have been misidentified as the single-channel correlate of the whole-cell N-type Ca2+ current in some previous studies.

Identification of mitogen-responsive ribosomal protein S6 kinase pp90rsk, a homolog of Xenopus S6 kinase II, in chicken embryo fibroblasts.

Antiserum raised against recombinant Xenopus ribosomal protein S6 kinase (rsk) was used to identify a 90,000-Mr ribosomal S6 kinase, pp90rsk, in chicken embryo fibroblasts. Adding serum to cells stimulated the phosphorylation of pp90rsk on serine and threonine residues and increased the activity of S6 kinase measured in immune complex assays. Xenopus S6 kinase II and chicken embryo fibroblast pp90rsk had nearly identical phosphopeptide maps.

Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase.

We have previously reported the isolation of cDNAs encoding two closely related Xenopus ribosomal S6 kinases, S6KII alpha and -beta (S. W. Jones, E. Erikson, J. Blenis, J. L. Maller, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 85:3377-3381, 1988). We report here the molecular cloning of one chicken and two mouse homologs of the Xenopus laevis cDNAs. As described for the Xenopus proteins, these cDNAs were found to predict polypeptides that contain two distinct kinase domains, of which one is most closely related to the catalytic subunit of cyclic AMP-dependent protein kinase and the other is most closely related to the catalytic subunit of phosphorylase b kinase. The three predicted proteins were more than 79% identical to the Xenopus S6KII alpha protein. The chicken and one of the mouse cDNAs were, respectively, 3.7 and 3.1 kilobase pairs in length, predicted proteins of 752 and 724 amino acids with molecular weights of 84.4 and 81.6 kilodaltons, and hybridized to mRNAs in fibroblasts and tissues of approximately 3.6 and 3.4 kilobases (kb). The second mouse cDNA was approximately 6.1 kilobase pairs and was not full length but predicted the C-terminal 633 amino acids of a protein that is similar to the C-terminal portion of Xenopus S6KII alpha. This clone hybridized to mRNA transcripts of 7.6 and 3.4 kb. In vitro transcription and translation of the chicken and the mouse cDNAs that predict complete proteins produced major products with apparent molecular weights of 96 and 84 kilodaltons. Analysis of mRNA levels in chicken tissues showed significant quantities of the 3.6-kb transcript in small and large intestine, spleen, and bursa. Both mouse cDNA were similarly expressed at significant levels in intestine, thymus, and lung; however, the 7.6-kb mRNA was differentially and more highly expressed in heart and brain. The two mouse cDNAs represent two different S6 kinase genes, as shown by comparison of their protein sequences, mRNA transcript sizes, genomic organizations, and nucleic acid sequences. We propose that this family of genes be named rsk, for ribosomal S6 kinase.

Structure-function studies of N-acyl derivatives of tryptophan that function as specific cholecystokinin receptor antagonists.

Benzotript, N-p-chlorobenzoyl-L-tryptophan, is a specific cholecystokinin receptor antagonist. In the present study we used dispersed pancreatic acini to examine tryptophan as well as several different N-acyl derivatives of tryptophan for their abilities to function as cholecystokinin receptor antagonists. L-Tryptophan, D-tryptophan as well as each acyl derivative tested inhibited cholecystokinin-stimulated amylase secretion and outflux of 45Ca and there was a good correlation between the ability of a particular agent to inhibit the action of cholecystokinin on acinar function and its ability to inhibit binding of 125I-labeled cholecystokinin to pancreatic acini. Results with butyloxycarbonyl-L-tryptophan indicated that the inhibition of the action of cholecystokinin caused by L-tryptophan and various acyl derivatives is specific, competitive and fully reversible. In functioning as a cholecystokinin receptor antagonist the relative potencies of the agents tested were: carbobenzoxy-L-tryptophan greater than benzotript greater than benzoyl-L-tryptophan = butyloxycarbonyl-L-tryptophan greater than acetyl-L-tryptophan greater than L-tryptophan. In inhibiting the actions of cholecystokinin, native as well as N-acyl derivatives of D-tryptophan were equipotent with the corresponding compound containing L-tryptophan. Although L-tryptophan inhibited the actions of cholecystokinin, L-phenylalanine, L-methionine or L-aspartic acid, even when tested at concentrations as high as 3 mM, did not alter the action of cholecystokinin on pancreatic acini. The antagonism of the actions of cholecystokinin was not restricted to N-acyl derivatives of L-tryptophan because butyloxycarbonyl-L-methionine and butyloxycarbonyl-L-phenylalanine but not butyloxycarbonyl-L-aspartic acid also antagonized the actions of cholecystokinin. These results demonstrate that both the nature of the N-acyl group and the amino acid residue are important determinants of the affinity of the antagonist for the cholecystokinin receptor. For derivatives of L-tryptophan, the more hydrophobic the N-acyl moiety, the greater the affinity of the derivative for the cholecystokinin receptor.

COOH-terminal fragments of cholecystokinin. A new class of cholecystokinin receptor antagonists.

COOH-terminal fragments of cholecystokinin varying in length from 1 to 3 amino acids and their NH2-terminal butyloxycarbonyl derivatives were investigated for their ability to interact with the cholecystokinin receptor on dispersed acini from guinea pig pancreas. No fragment stimulated amylase secretion when present alone, but each of the butyloxycarbonyl derivatives and the COOH-terminal tripeptide amide inhibited the stimulation of enzyme secretion by cholecystokinin. In each case the inhibition was surmounted by increasing the concentration of cholecystokinin. Each fragment also inhibited binding of 125I-labeled cholecystokinin, with significant inhibition occurring with 30 microM butyloxycarbonyl tripeptide amide, 0.3 mM butyloxycarbonyl dipeptide amide, 10 mM butyloxycarbonyl phenylalanine amide and 3 mM tripeptide amide of cholecystokinin. In each case, there was a close correlation between the ability of the fragment to inhibit binding of 125I-labeled cholecystokinin and its ability to inhibit cholecystokinin-stimulated amylase release, cholecystokinin-stimulated 45Ca outflux and cholecystokinin-stimulated residual stimulation of amylase secretion. The inhibition of amylase secretion caused by the butyloxycarbonyl tripeptide of cholecystokinin was reversible and specific for those peptides which interact with the cholecystokinin receptor (i.e., cholecystokinin, caerulein, gastrin); it did not inhibit the actions of bombesin, carbachol, physalaemin, vasoactive intestinal peptide, secretin, PHI, ionophore A23187 or 8-bromo cyclic AMP. These results demonstrate that COOH-terminal fragments of cholecystokinin comprise a new class of cholecystokinin receptor antagonists.

Interaction of substance P antagonists with substance P receptors on dispersed pancreatic acini.

In the present study we examined the abilities of three analogs of substance P, [D-Pro2-, D-Phe7-, D-Trp9]-substance P, [D-Pro2-, D- Trp7 ,9]-substance P and [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to alter substance P-induced changes in pancreatic acinar cell function and to occupy substance P receptors. At 30 microM, each analog of substance P lacked agonist activity and inhibited amylase secretion stimulated by substance P receptor agonists. The inhibition was reversible and specific for peptides that interact with substance P receptors (physalaemin, substance P, eledoisin, kassinin ). The analogs of substance P did not inhibit the actions of cholecystokinin, caerulein, gastrin, carbamylcholine, secretin, vasoactive intestinal peptide, PHI, ionophore A23187 or 8Br -cAMP. At high concentrations, [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P, but not [D-Pro2-, D- Trp7 ,9]-substance P or [D-Pro2-, D-Phe7-, D-Trp9]-substance P, caused a small but significant inhibition of bombesin-stimulated amylase release. For each analog of substance P, the inhibition was competitive in nature in that there was a rightward shift of the dose-response curve for physalaemin-stimulated amylase secretion with no change in efficacy. From Schild plots of the ability of [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P to inhibit either substance p- or physalaemin-stimulated amylase release, the slopes were not different from unity. For each analog of substance P, there was a close correlation between its ability to inhibit substance P- or physalaemin-stimulated amylase release and its ability to inhibit binding of 125I-labeled substance P or 125I-labeled physalaemin. [D-Arg1-, D-Pro2-, D- Trp7 ,9-, Leu11 ]-substance P was 2-fold more potent than [D-Pro2-, D- Trp7 ,9]-substance P which was 4-fold more potent than [D-Pro2-, D-Phe7-, D-Trp9]-substance P, (i.e., pA2 6.1, 5.9, and 5.2, respectively). For each analog, the dose-response curve for its ability to inhibit physalaemin-stimulated amylase release was superimpossible on the dose-response curve for its ability to inhibit binding of 125I-labeled physalaemin. These results indicate that each of these analogs of substance P is a specific competitive inhibitor of the action of the substance P on dispersed acini from guinea-pig pancreas, and that their abilities to inhibit substance P-induced changes in acinar cell function can be accounted for by their abilities to occupy the substance P receptor.

Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action.

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.

Surface charge and calcium channel saturation in bullfrog sympathetic neurons.

Currents carried by Ba2+ through calcium channels were recorded in the whole-cell configuration in isolated frog sympathetic neurons. The effect of surface charge on the apparent saturation of the channel with Ba2+ was examined by varying [Ba2+]o and ionic strength. The current increased with [Ba2+]o, and the I-V relation and the activation curve shifted to more positive voltages. The shift of activation could be described by Gouy-Chapman theory, with a surface charge density of 1 e-/140 A2, calculated from the Grahame equation. Changes in ionic strength (replacing N-methyl-D-glucamine with sucrose) shifted the activation curve as expected for a surface charge density of 1 e-/85 A2, in reasonable agreement with the value from changing [Ba2+]o. The instantaneous I-V for fully activated channels also changed with ionic strength, which could be described either by a low surface charge density (less than 1 e-/1,500 A2), or by block by NMG with Kd approximately 300 mM (assuming no surface charge). We conclude that the channel permeation mechanism sees much less surface charge than the gating mechanism. The peak inward current saturated with an apparent Kd = 11.6 mM for Ba2+, while the instantaneous I-V saturated with an apparent Kd = 23.5 mM at 0 mV. This discrepancy can be explained by a lower surface charge near the pore, compared to the voltage sensor. After correction for a surface charge near the pore of 1 e-/1,500 A2, the instantaneous I-V saturated as a function of local [Ba2+]o, with Kd = 65 mM. These results suggest that the channel pore does bind Ba2+ in a saturable manner, but the current-[Ba2+]o relationship may be significantly affected by surface charge.

Calcium currents in bullfrog sympathetic neurons. I. Activation kinetics and pharmacology.

The calcium current of bullfrog sympathetic neurons activates and deactivates rapidly (tau less than 3 ms). For brief depolarizations, the current can be fit reasonably well by a Hodgkin-Huxley-type model with a single gating particle of charge +3. With 2 mM Ca2+ as the charge carrier, half-maximal activation occurs at approximately -5 mV, near the voltage where activation and deactivation are slowest. When extracellular divalent ion concentrations are reduced, monovalent ions (e.g., Na+ and methylammonium) produce kinetically similar inward currents. Current carried by Ba2+ is blocked by Cd2+ at micromolar concentrations, and by 100 nM omega-conotoxin. Commercially available saxitoxin blocks the current, but different batches have quantitatively different potency. The dihydropyridine agonist Bay K 8644 induces a slight shift in activation kinetics to more negative voltages, with little effect on the peak current. Nifedipine at least partially reverses the effect of Bay K 8644, but has little effect on its own. Muscarinic agonists and other ligands that inhibit the M-type potassium current of frog sympathetic neurons have weak inhibitory effects on the calcium current as well. One interpretation of these results is that the N-type calcium current predominates in these cells, with a minor contribution of L-type current.

Calcium currents in bullfrog sympathetic neurons. II. Inactivation.

Calcium currents in bullfrog sympathetic neurons inactivate slowly and partially during depolarizations lasting 0.5-1 s. There is also a slower (minutes) inactivation process with a broad voltage dependence. An irreversible loss of current (rundown) is prominent with low concentrations of intracellular Ca2+ buffers, with either Ca2+ or Ba2+ as the charge carrier. The extent and rate of the more rapid inactivation process are maximal near the voltage at which the peak inward current is generated, suggesting that inactivation might be Ca2+ dependent. However, inactivation occurs with either Ca2+ or Ba2+ as the charge carrier, is not prevented by strong buffering of intracellular Ca2+ with 10 mM BAPTA, and varies little as the peak current is changed 10-fold by changing the divalent ion concentration. That is, rapid inactivation is not explained by simple versions of voltage, Ca2+- or current-dependent inactivation models. A model in which ion binding within the channel allows a slower, rate-limiting inactivation process fits some but not all of the observed features of inactivation. A purely voltage-dependent three-state cyclic model fits the data if microscopic inactivation is favored by hyperpolarization.