Peak Calcium Levels Research Articles

Etelcalcetide use During Maintenance Hemodialysis and Incidence of Parathyroidectomy After Kidney Transplantation

Etelcalcetide is an i.v. calcimimetic agent, effectively reducing parathyroid hormone levels in patients on maintenance hemodialysis (HD). The clinical impact of discontinuing etelcalcetide at the time of kidney transplantation is unknown. We retrospectively reviewed all patients on HD meeting predefined criteria who received a kidney transplant at our institution between January 1, 2015, and December 12, 2022. The incidence of parathyroidectomy and the evolution of calcium, phosphate, and intact parathyroid hormone (iPTH) levels after transplantation was analyzed according to the type of calcimimetic treatment before transplantation (cinacalcet vs. etelcalcetide vs. none). Overall, 372 patients (aged 53 years; interquartile range [IQR]: 42-62 years) were included. At the time of transplantation, 35, 75, and 262 patients were under etelcalcetide, cinacalcet, or no calcimimetic, respectively. After 1064 (IQR: 367-1658) days, the incidences of parathyroidectomy in the etelcalcetide, cinacalcet, no calcimimetic groups were 29%, 12%, and 1%, respectively (P< 0.001). Etelcalcetide was associated with an increased incidence of parathyroidectomy after adjustment for age, sex, and HD vintage (hazard ratio [HR]: 97.0, 95% confidence interval [CI]: 19.1-493.9, P< 0.001). The incidence of parathyroidectomy was related to etelcalcetide dosage (6/11 [54.6%] in patients with≥ 10 mg vs. 4/24 [16.7%] in patients with< 10 mg, P= 0.02). Moreover, peak calcium levels were higher (P< 0.001) and parathyroidectomy was performed earlier (median 80 vs. 480 days, P< 0.001) in the etelcalcetide compared with the cinacalcet group. Long-term graft function, graft loss, and mortality were similar. Etelcalcetide use during maintenance HD is associated with an increased incidence of early parathyroidectomy after transplantation compared to cinacalcet or no calcimimetic.

WTP2.4 Can we predict parathyroid adenoma size preoperatively with biochemical markers?

Abstract Aim In primary hyperparathyroidism, parathyroidectomy remains the mainstay of treatment. However, localising abnormal parathyroid glands can often prove challenging, and re-operations may be necessary. We aim to evaluate any correlation that might exist between parathyroid hormone (PTH) levels, preoperative peak corrected calcium levels and cinacalcet use with adenoma size. Methods We retrospectively reviewed consecutive parathyroidectomies performed for primary hyperparathyroidism by two experienced upper gastrointestinal surgeons (VS and PT) in a teaching hospital between April 2013 and December 2022. Hospital database was accessed to obtain data for biochemical markers, operation notes and histopathology reports. Spearman correlation coefficient was computed to evaluate linear relationship between PTH, peak calcium levels and adenoma size. Mann-Whitney-U test was used to compare cinacalcet use and adenoma sizes. P-value&amp;lt;0.05 was statistically significant. Results 170 parathyroidectomies for primary hyperparathyroidism were performed during the study period. Patient cohort was predominantly female (n=131, 77.1%). Mean age of study population was 58±12 years. There were positive correlations between adenoma size and preoperative peak corrected calcium levels (R=0.25, p=0.001) and PTH levels (R=0.4, p&amp;lt;0.001) respectively. In patients who took cinacalcet preoperatively (n=46, 27.1%), there was an association with larger adenoma size (18±8mm vs 22±12mm, p=0.009). Conclusion Higher preoperative PTH levels and peak corrected calcium levels along with cinacalcet use may be associated with larger adenoma sizes. Further studies are required to validate these findings.

Abstract #1407860: Rare Cause of Non-PTH Mediated Hypercalcemia Due to Pneumocystis Pneumonia in Renal Transplant Recipient

Hypercalcemia secondary to persistent hyperparathyroidism is common in kidney transplant recipients (KTR). We report a rare cause of non-PTH mediated hypercalcemia in KTR due to pneumocystis jirovecii pneumonia (PJP). The incidence of PJP in KTR is 5-15% in absence of antibiotic prophylaxis. Incidence of hypercalcemia in KTR with PJP is unclear, but scattered case reports are found in literature. The proposed mechanism for hypercalcemia is an infectious granulomatous reaction. PJP induced hypercalcemia needs to be considered in KTR patients with non-PTH mediated hypercalcemia. A 62-year-old male with kidney transplant for renal failure from glomerulosclerosis, three years prior to presentation was admitted for dyspnea and fever. Imaging showed bilateral lung infiltrates, concerning for multifocal infection. Considering his imaging findings and elevated Beta D glucan levels, he was empirically started on PJP treatment with atovaquone on day 4 of admission. Bactrim was not used due to acute renal failure. On day 1, calcium was 10 mg/dL (8.4-10.4), which then progressively increased from 10.7mg/dl on day 4 to a peak calcium level of 14.4 mg/dL on day 12. Further work up showed PTH: 4.7 pg/mL (12-88), PTHrP: 0.6 pMol/L (< /= 4.2), Vitamin D 1, 25: 198 pg/mL (18-72), Vitamin D 25: 79 ng/mL (20-100), SPEP: no M spike, UPEP: no M spike but had a lysozyme band suggestive of granulomatous infection, other fungal studies negative, normal thyroid levels, AFB culture negative. On day 12, he was started on prednisone 60mg presuming hypercalcemia from PJP. On day 13, calcium improved to 11.7 mg/ dL, but his renal function worsened requiring hemodialysis (HD). On day 14, he had respiratory failure requiring intubation and underwent bronchio-alveolar lavage (BAL) which was positive for pneumocystis PCR. He was started on bactrim on day 17 and continued on high dose steroid. By day 19, he was extubated, renal function stabilized without HD and calcium normalized to 8.9 mg/dL. Unfortunately, he had gastrointestinal bleed and refused further procedures eventually opting for hospice on day 23. PJP is a common opportunistic infection in immunosuppressed patients like KTR. The advent of prophylaxis has significantly decreased the incidence of PJP in KTR to 0.3%-2.6%. PJP in KTR is sometimes associated with hypercalcemia. Prevalence of hypercalcemia in KTR with PJP is unclear, but as per a hospital based retrospective study (Hamroun et al., 2019), 37% of PJP patients in KTR (18/49) had hypercalcemia that preceded pneumonia/findings on imaging. The proposed mechanism for hypercalcemia is granulomatous in nature, due to increased extra renal production of 1 alpha hydroxylase enzyme from increased macrophage recruitment in lungs to clear pneumocystis organisms. Hypercalcemia resolved after completion of treatment and resolution of PJP. High dose steroids can be used as hypercalcemia is mediated by a granulomatous reaction. It is crucial for physicians to have a high index of suspicion for PJP as the etiology of pneumonia in immunocompromised organ transplant recipients with non- PTH mediated hypercalcemia, thus ensuring timely diagnosis and treatment.

Spine head calcium as a measure of summed postsynaptic activity for driving synaptic plasticity.

We use a computational model of a hippocampal CA1 pyramidal cell to demonstrate that spine head calcium provides an instantaneous readout at each synapse of the postsynaptic weighted sum of all presynaptic activity impinging on the cell. The form of the readout is equivalent to the functions of weighted, summed inputs used in neural network learning rules. Within a dendritic layer, peak spine head calcium levels are either a linear or sigmoidal function of the number of coactive synapses, with nonlinearity depending on the ability of voltage spread in the dendrites to reach calcium spike threshold. This is strongly controlled by the potassium A-type current, with calcium spikes and the consequent sigmoidal increase in peak spine head calcium present only when the A-channel density is low. Other membrane characteristics influence the gain of the relationship between peak calcium and the number of active synapses. In particular, increasing spine neck resistance increases the gain due to increased voltage responses to synaptic input in spine heads. Colocation of stimulated synapses on a single dendritic branch also increases the gain of the response. Input pathways cooperate: CA3 inputs to the proximal apical dendrites can strongly amplify peak calcium levels due to weak EC input to the distal dendrites, but not so strongly vice versa. CA3 inputs to the basal dendrites can boost calcium levels in the proximal apical dendrites, but the relative electrical compactness of the basal dendrites results in the reverse effect being less significant. These results give pointers as to how to better describe the contributions of pre- and postsynaptic activity in the learning "rules" that apply in these cells. The calcium signal is closer in form to the activity measures used in traditional neural network learning rules than to the spike times used in spike-timing-dependent plasticity.

Dendritic diameters affect the spatial variability of intracellular calcium dynamics in computer models.

There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio (SVR) of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented.

Abstract 159: Ablation of the Hypertension Candidate Gene ATP2B1 Leads To Deficient Calcium Cycling, Systolic Dysfunction and Heart Failure Following Pressure Overload

Mutations in ATP2B1 encoding the ubiquitous calcium extrusion pump Plasma Membrane Calcium ATPase 1 (PMCA1) have recently identified it as having the strongest association of any gene to hypertension, yet the role of PMCA1 in the pressure-overloaded heart is not known. To investigate this we generated a novel mouse line carrying cardiomyocyte-specific deletion of PMCA1 (PMCA1 cko ) and challenged them with transverse aortic constriction (TAC) alongside littermate ‘floxed’ controls (PMCA1 f/f ). After two weeks, echocardiographic analysis revealed signs of systolic dysfunction and left ventricular (LV) dilation in PMCA1 cko hearts as evidenced by reduced fractional shortening and increased diastolic diameter (both p&lt;0.05), whilst function in PMCA1 f/f TAC controls remained preserved. This was accompanied by an increase in normalised lung weight in PMCA1 cko mice compared to sham operated and TAC controls (p&lt;0.05) indicative of pulmonary congestion and a progression into LV failure, despite comparable hypertrophic growth amongst the two TAC cohorts. Hemodynamic analysis following LV catheterisation revealed contractility, as measured by left ventricular elastance (E es ), to be increased in controls after TAC (PMCA1 f/f TAC 12.69 ± 1.63 vs sham 7.02 ± 1.11 mmHg/μl, p&lt;0.05), a change which was not reciprocated in knockout hearts (PMCA1 cko TAC 7.70 ± 1.19 vs sham 7.22 ± 1.55 mmHg/μl). To examine whether altered calcium handling could be the underlying cause of the observed phenotype, cardiomyocytes were isolated following one week TAC and loaded with Indo-1, prior to the onset of failure in PMCA1 cko hearts. Compatible with an increase in E es , systolic calcium levels were higher in PMCA1 f/f myocytes following pressure overload compared to sham controls (p&lt;0.05), whilst PMCA1 cko TAC myocytes displayed equivalent peak calcium levels to their respective sham controls. These results suggest that PMCA1 may play a necessary role in enhancing calcium cycling during the early response to pressure overload, and that disrupting this gene may increase the susceptibility to heart failure under these conditions. This may provide first evidence of a novel genetic basis for the development of heart failure in a proportion of hypertensive patients.

A computational study of the influence of synaptic cooperativity on synaptic plasticity in a hippocampal CA1 pyramidal cell

We use a computational model to investigate the possible cooperativity between coactive synapses, both within and between input pathways, in determining plasticity outcomes at individual synapses on a hippocampal CA1 pyramidal cell (PC). The detailed compartmental model of the PC, based on a reconstructed morphology, contains distributions of core ion channels that control voltage transients and calcium entry in the soma and dendrites. Of particular note are the increasing density of A-type potassium channels (KA) with distance in the apical dendrites that provide strong filtering of voltage transients, and high-voltage-gated calcium channels (VGCC) in the dendrites and spines that may contribute to calcium entry in spine heads. Groups of spatially diffuse or clustered excitatory synapses on spines in dendritic layers stratum lacunosum-moleculare (SLM), stratum radiatum (SR) and stratum oriens (SO) are stimulated with single spikes or high frequency bursts. Synaptic currents are mediated by AMPA and NMDA receptors. A proportion of the NMDAR-mediated current is carried by calcium ions. As an indicator of likely synaptic plasticity, the peak calcium level (pCa) obtained in each spine head is measured. Figure ​Figure11 shows results from typical simulations. Peak calcium initially is a somewhat linear function of the number of synchronously active synapses, until saturation. The gain is higher if the density of KA is reduced. With VGCCs, pCa becomes a nonlinear, sigmoidal function. Figure 1 Average spine head peak calcium due to single synchronous stimulation of different numbers of synapses in SLM, in four cell configurations (1) high (standard) KA, (2) high KA with VGCC, (3) low (one third) KA, (4) with VGCC. Error bars show standard deviation. ... Other results show that presynaptic burst stimulation is much more effective at raising spine head calcium levels than single stimuli. Similarly, small numbers of coactive synapses on a single oblique dendrite can achieve high pCa. Activity in one layer, such as SLM can affect pCa in another layer (SR) but only if KA is reduced. In summary, burst stimulation and colocalised activity are most likely to lead to high pCa and hence LTP. Cooperativity across dendritic pathways is possible, but needs specific cellular and stimulation conditions.

Spine Calcium Transients Induced by Synaptically-Evoked Action Potentials Can Predict Synapse Location and Establish Synaptic Democracy

CA1 pyramidal neurons receive hundreds of synaptic inputs at different distances from the soma. Distance-dependent synaptic scaling enables distal and proximal synapses to influence the somatic membrane equally, a phenomenon called “synaptic democracy”. How this is established is unclear. The backpropagating action potential (BAP) is hypothesised to provide distance-dependent information to synapses, allowing synaptic strengths to scale accordingly. Experimental measurements show that a BAP evoked by current injection at the soma causes calcium currents in the apical shaft whose amplitudes decay with distance from the soma. However, in vivo action potentials are not induced by somatic current injection but by synaptic inputs along the dendrites, which creates a different excitable state of the dendrites. Due to technical limitations, it is not possible to study experimentally whether distance information can also be provided by synaptically-evoked BAPs. Therefore we adapted a realistic morphological and electrophysiological model to measure BAP-induced voltage and calcium signals in spines after Schaffer collateral synapse stimulation. We show that peak calcium concentration is highly correlated with soma-synapse distance under a number of physiologically-realistic suprathreshold stimulation regimes and for a range of dendritic morphologies. Peak calcium levels also predicted the attenuation of the EPSP across the dendritic tree. Furthermore, we show that peak calcium can be used to set up a synaptic democracy in a homeostatic manner, whereby synapses regulate their synaptic strength on the basis of the difference between peak calcium and a uniform target value. We conclude that information derived from synaptically-generated BAPs can indicate synapse location and can subsequently be utilised to implement a synaptic democracy.

The Positive Force-Frequency Relationship Is Maintained In Absence Of Sarcoplasmic Reticulum Function In Rabbit, But Not In Rat Myocardium

Myocardial calcium handling differs between species, mainly in the relative contribution between the sources for activator calcium. To investigate the role of the myofilaments and intracellular calcium decline in governing the relaxation phase of cardiac muscle, and to elucidate additional determinants of relaxation other than the sarcoplasmic reticulum (SR) at various frequencies within the in vivo range, the present study was performed by altering the calcium handling in rat and rabbit. Trabeculae, iontophoretically loaded with bis-fura-2 to monitor cytoplasmic calcium levels, were subjected to ryanodine and cyclopiazonic acid to inhibit SR function. Simultaneous force and [Ca2+]i measurements were obtained at 1–4 Hz in rabbit and at 4–8 Hz in rat before and after SR inhibition. Inhibition of the SR resulted in increased diastolic and peak calcium levels as well as decreased developed force in both species. Calcium transient amplitude decreased in rat, but increased in rabbit after SR inhibition. Time to peak tension, time from peak tension to 50% relaxation, time to peak calcium, and time from peak calcium to 50% calcium decline were all prolonged. Results suggest that L-type calcium channel current is responsible for increases in calcium with increasing frequency, and that the SR amplifies this effect in response to increased L-type current. The response of the myofilaments to alterations in calcium handling plays a critical role in the final determination of force, and may differ between species. These results imply the balance between force relaxation and calcium decline is significantly different in larger mammals, necessitating a critical re-evaluation of how myocardial relaxation is governed, specifically regarding frequency-dependent activation.

The positive force–frequency relationship is maintained in absence of sarcoplasmic reticulum function in rabbit, but not in rat myocardium

Myocardial calcium handling differs between species, mainly in the relative contribution between the sources for activator calcium. To investigate the role of the myofilaments and intracellular calcium decline in governing the relaxation phase of cardiac muscle, and to elucidate additional determinants of relaxation other than the sarcoplasmic reticulum (SR) at various frequencies within the in vivo range, the present study was performed by altering the calcium handling in rat and rabbit. Trabeculae, iontophoretically loaded with bis-fura-2 to monitor cytoplasmic calcium levels, were subjected to ryanodine and cyclopiazonic acid to inhibit SR function. Simultaneous force and [Ca(2+)](i) measurements were obtained at 1-4 Hz in rabbit and at 4-8 Hz in rat before and after SR inhibition. Inhibition of the SR resulted in increased diastolic and peak calcium levels as well as decreased developed force in both species. Calcium transient amplitude decreased in rat, but increased in rabbit after SR inhibition. Time to peak tension, time from peak tension to 50% relaxation, time to peak calcium, and time from peak calcium to 50% calcium decline were all prolonged. Results suggest that L-type calcium channel current is responsible for increases in calcium with increasing frequency, and that the SR amplifies this effect in response to increased L-type current. The response of the myofilaments to alterations in calcium handling plays a critical role in the final determination of force, and may differ between species. These results imply the balance between force relaxation and calcium decline is significantly different in larger mammals, necessitating a critical re-evaluation of how myocardial relaxation is governed, specifically regarding frequency-dependent activation.

A lipoxygenase product, hepoxilin A 3, enhances nerve growth factor-dependent neurite regeneration post-axotomy in rat superior cervical ganglion neurons in vitro

Hepoxilins are 12-lipoxygenase metabolites of arachidonic acid found in the CNS. They can modulate neuronal signaling but their functions are not known. We examined the effects of hepoxilin A 3 on neurite outgrowth post-axotomy in an in vitro model of spinal cord transection using superior cervical ganglion neurons. In the absence of nerve growth factor, hepoxilin A 3 did not support neuronal survival, or regeneration post-axotomy but did significantly enhance neurite regeneration in the presence of nerve growth factor. As early as 1 h post-injury hepoxilin A 3-treated cultures (+nerve growth factor) had significantly more neurites than controls (nerve growth factor alone). Average hourly rates of outgrowth in hepoxilin A 3-treated cultures were significantly higher than in controls for at least 12 h post-injury, suggesting that the effect of hepoxilin A 3 is maintained in vitro for several hours post-injury. In uninjured neurons hepoxilin A 3 caused a rapid but transient increase in intracellular calcium in the somata; by 2 min post-addition, calcium levels decreased to a new stable plateau significantly higher than pre treatment levels. In injured neurons, hepoxilin A 3 addition immediately post-transection caused a rapid transient increase in intracellular calcium in cell bodies; however, peak calcium levels were significantly lower than in uninjured neurons and the new baseline lower than in uninjured cells. In uninjured cells hepoxilin A 3 addition in zero calcium produced the same pattern, a transient elevation and subsequent decline to a new stable baseline significantly above rest but in injured cells levels fell rapidly to pretreatment values. Taken overall, these findings demonstrate a novel role for hepoxilins as a potentiator of neurite regeneration. They also provide the first evidence that this lipoxygenase metabolite can alter intracellular calcium in neurons by causing release of calcium from intracellular stores and modulating calcium influx mechanisms.

The Inositol Trisphosphate Receptor Regulates a 50-Second Behavioral Rhythm in C. elegans

The C. elegans defecation cycle is characterized by the contraction of three distinct sets of muscles every 50 s. Our data indicate that this cycle is regulated by periodic calcium release mediated by the inositol trisphosphate receptor (IP 3 receptor). Mutations in the IP 3 receptor slow down or eliminate the cycle, while overexpression speeds up the cycle. The IP 3 receptor controls these periodic muscle contractions nonautonomously from the intestine. In the intestinal cells, calcium levels oscillate with the same period as the defecation cycle and peak calcium levels immediately precede the first muscle contraction. Mutations in the IP 3 receptor slow or eliminate these calcium oscillations. Thus, the IP 3 receptor is an essential component of the timekeeper for this cycle and represents a novel mechanism for the control of behavioral rhythms.

Elevated basal and thapsigargin-stimulated intracellular calcium of platelets and lymphocytes from bipolar affective disorder patients measured by a fluorometric microassay

Background: A number of investigators have reported finding elevated basal and stimulated intracellular calcium levels in the platelets or lymphocytes of bipolar disorder patients. Methods: Intracellular calcium was measured by a micro fura-2 fluorometric method in the platelets and lymphocytes of 30 affective disorder patients and 14 control subjects. Results: We observed significantly elevated basal calcium concentrations in bipolar patient platelets and lymphocytes compared to control subjects. Bipolar patient platelet calcium responses to thrombin, serotonin, and thapsigargin were also significantly greater than control subjects. The peak calcium levels of lymphocytes of bipolar patients were greater than control subjects only when stimulated by thapsigargin. There were significant differences between bipolar and unipolar patients in basal and thapsigargin-stimulated calcium measures but not between bipolar I and bipolar II patients. Unmedicated versus medicated calcium measures were not significantly different. We also found little correlation between calcium measures and the severity of mood rating. Conclusions: Using this method, we were able to confirm and extend the work of others, indicating altered intracellular calcium homeostasis in the blood cells of bipolar disorder patients. In addition, our data suggest that storage operated calcium channels may be the source of the elevated intracellular calcium in platelets and lymphocytes of bipolar patients.

Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes.

1. The mechanisms underlying hemispheric asymmetry of the inositol 1, 4,5-trisphosphate (InsP3)-calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium-activated Cl- currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3. 2. The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere. 3. Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere. 4. Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70 % greater in the animal hemisphere. 5. Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 micro m compared with 2.25 micro m in the vegetal hemisphere. 6. The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl- channels.

Clinical evaluation of total serum calcium in primary hyperparathyroidism and the risk of death after surgery.

This study aims to understand the behaviour and clinical value of total serum calcium in untreated primary hyperparathyroidism, to identify the significant relationship between pre-operative total serum calcium and the risk of death after surgery, and to consider the issue of when to operate. The risk of death after surgery was studied as dependent on pre-operative serum calcium levels in a series of 896 patients followed up for mean 12.9 years (SD 6.1) after surgery. The predictive power of pre-operative peak calcium levels was stronger than that of mean calcium levels. It was found that an increase of peak serum calcium from 2.60 mmolL-1 to 2.90 in one patient meant a death risk increase, with 38% still 5 years after surgery. The marginal risk increase per mmol L-1 was found to be higher below the peak serum calcium level of 2.90 mmol L-1 than above that level. The variation of total serum calcium before surgery was found to be substantial and the occurrence of transient high serum calcium levels was not unusual. Therefore, conservative surveillance with yearly total serum calcium estimations seems insufficient. Rather, early surgery, when serum calcium levels are not more than moderately increased, appears to be the most favourable alternative.

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent "amplification" was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high-NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel natural inhibitor from Candida albicans hyphae causing dissociation of the neutrophil respiratory burst response to chemotactic peptides from other post-activation events.

Previous work established that Candida albicans hyphae release several inhibitors of human neutrophil function. We now report that the crude hyphal inhibitory product (CHIP) inhibits superoxide anion (O2-) production stimulated by FMLP in a dose-related manner with an EC50 of approximately 2 micrograms/ml. CHIP also inhibited O2- production stimulated by A23187 and by opsonized zymosan, although this effect could be overcome by increasing the concentration of agonist. No inhibition of the PMA-stimulated burst was seen at any concentration of PMA tested, indicating that CHIP neither affected polymorphonuclear neutrophil viability nor quenched superoxide anion detection. A saturating dose of inhibitor had no effect on chemotaxis stimulated either by 0.1 to 100 nM FMLP or by zymosan-activated serum. Peak inositol trisphosphate levels stimulated by FMLP were not inhibited by a dose of CHIP producing maximum inhibition of FMLP-induced superoxide production. Peak changes in cytosolic free calcium levels (as measured by indo-1 fluorescence) stimulated by 50 nM or greater FMLP were unaffected by CHIP, although for subsaturating doses of FMLP a more rapid decline from peak calcium levels was seen in CHIP-exposed cells. Taken together, these data suggest that the common fungal pathogen C. albicans releases a substance that selectively impairs the neutrophil respiratory burst. It appears to do so without inhibiting the fully assembled NADPH oxidase and with minimal or no effect on events tightly coupled to FMLP-R/G protein activation, suggesting that these events may be uncoupled from activation of the burst. In addition, the absence of effect of CHIP on chemotaxis despite profound inhibition of the respiratory burst suggests these neutrophil functions may be mediated by divergent transduction pathways.