Epidemiology, clinical profile, management, and outcome of COVID-19-associated rhino-orbital-cerebral mucormycosis in 2826 patients in India - Collaborative OPAI-IJO Study on Mucormycosis in COVID-19 (COSMIC), Report 1.

Purpose: COVID-19-associated rhino-orbital-cerebral mucormycosis (ROCM) has reached epidemic proportion during India's second wave of COVID-19 pandemic, with several risk factors being implicated in its pathogenesis. This study aimed to determine the patient demographics, risk factors including comorbidities, and medications used to treat COVID-19, presenting symptoms and signs, and the outcome of management. Methods: This was a retrospective, observational study of patients with COVID-19-associated ROCM managed or co-managed by ophthalmologists in India from January 1, 2020 to May 26, 2021. Results: Of the 2826 patients, the states of Gujarat (22%) and Maharashtra (21%) reported the highest number of ROCM. The mean age of patients was 51.9 years with a male preponderance (71%). While 57% of the patients needed oxygen support for COVID-19 infection, 87% of the patients were treated with corticosteroids, (21% for > 10 days). Diabetes mellitus (DM) was present in 78% of all patients. Most of the cases showed onset of symptoms of ROCM between day 10 and day 15 from the diagnosis of COVID-19, 56% developed within 14 days after COVID-19 diagnosis, while 44% had delayed onset beyond 14 days. Orbit was involved in 72% of patients, with stage 3c forming the bulk (27%). Overall treatment included intravenous amphotericin B in 73%, functional endoscopic sinus surgery (FESS)/paranasal sinus (PNS) debridement in 56%, orbital exenteration in 15%, and both FESS/PNS debridement and orbital exenteration in 17%. Intraorbital injection of amphotericin B was administered in 22%. At final follow-up, mortality was 14%. Disease stage >3b had poorer prognosis. Paranasal sinus debridement and orbital exenteration reduced the mortality rate from 52% to 39% in patients with stage 4 disease with intracranial extension (p < 0.05). Conclusion: : Corticosteroids and DM are the most important predisposing factors in the development of COVID-19-associated ROCM. COVID-19 patients must be followed up beyond recovery. Awareness of red flag symptoms and signs, high index of clinical suspicion, prompt diagnosis, and early initiation of treatment with amphotericin B, aggressive surgical debridement of the PNS, and orbital exenteration, where indicated, are essential for successful outcome.

Correction of preexisting astigmatism by penetrating arcuate keratotomy in femtosecond laser-assisted cataract surgery.

Purpose: To evaluate the astigmatism correcting effect of penetrating arcuate keratotomy (AK) done during femtosecond laser-assisted cataract surgery (FLACS). Methods: In this nonrandomized prospective study, 80 eyes of 70 patients were studied. The study included patients who underwent combined FLACS and AK, with corneal astigmatism ranging from 0.4 to 1.5 diopters (D). Femtosecond laser-assisted penetrating arcuate keratotomies were created at 8 mm optical zone at 80% depth and were centered at the limbus. Keratometric astigmatism was measured prior to and 3 months post-surgery. Vector analysis was performed using Power vector analysis method. Results: The mean preoperative keratometric astigmatism without accounting for axis was 0.85 ± 0.27 D, which reduced significantly to 0.47 ± 0.27 D at 3-month follow-up. The mean astigmatism correction attained without accounting for axis was 0.38 ± 0.32 D. The vector corrected mean preoperative astigmatism was 0.85 ± 0.27 D which reduced significantly to 0.50 ± 0.31 D postoperatively (P < 0.001, 95% CI). Vector corrected mean astigmatism correction attained was 0.35 ± 0.38 D. There were no significant intraoperative or postoperative complications. Conclusion: Preexisting astigmatism can be tackled effectively with penetrating AK during FLACS although under correction is observed with present nomograms. Further refinements may achieve better correction.

All India Ophthalmological Society - Oculoplastics Association of India consensus statement on preferred practices in oculoplasty and lacrimal surgery during the COVID-19 pandemic.

Oculoplastic surgeries encompass both emergency surgeries for traumatic conditions and infectious disorders as well as elective aesthetic procedures. The COVID-19 pandemic has brought about a drastic change in this practice. Given the highly infectious nature of the disease as well as the global scarcity of medical resources; it is only prudent to treat only emergent conditions during the pandemic as we incorporate evidence-based screening and protective measures into our practices. This manuscript is a compilation of evidence-based guidelines for surgical procedures that oculoplastic surgeons can employ during the COVID-19 pandemic. These guidelines also serve as the basic framework upon which further recommendations may be based on in the future, as elective surgeries start being performed on a regular basis.

Isolated bilateral uveitis in child with hypereosinophilic syndrome.

The ocular involvement has rarely been described in hypereosinophilic syndrome (HES). We report an 8-year-old girl with HES and isolated bilateral uveitis as end-organ damage. Almost 20 months after detection of persistent asymptomatic eosinophilia, she developed complete loss of vision in right eye due to retinal detachment and decreased vision in left eye. We treated this organ-threatening condition with prednisolone and imatinib mesylate, although she was negative for FIP1L1-PDGRFA fusion gene. The vision in her left eye returned to normal. At present, the child is on alternate-day low-dose prednisolone and daily imatinib. Early recognition and aggressive treatment is essential in HES with ocular involvement to save vision. Imatinib is a useful adjuvant drug even in PDGRFA/FIP1L1-negative HES.

Antioxidants and vision health: facts and fiction.

A number of nutritional supplements containing antioxidants are advertised for better vision health. Do they benefit the average consumer? The literature was examined for the effectiveness of antioxidants for human eye health, and for the intricacies in collection of such evidence. The following diseases were considered: cataract, glaucoma, age-related macular degeneration (AMD), retinopathy, retinitis pigmentosa, eye infections, and uveitis. The literature indicates that antioxidant supplements plus lutein have a reasonable probability of retarding AMD. For glaucoma, such supplements were ineffectual in some studies but useful in others. In some studies, antioxidant rich fruits and vegetables were also useful for protection against glaucoma. For diabetic retinopathy, antioxidant supplements may have a small benefit, if any, but only as an adjunct to glycemic control. In very high-risk premature retinopathy and retinitis pigmentosa, antioxidant supplements may be beneficial but those with excess Vitamin E should be avoided. For cataract, there is no evidence for an advantage of such nutritional supplements. However, lubricant drops containing N-acetylcarnosine may be helpful in initial stages of the disease. For eye infections and other causes of uveitis, antioxidants have not been found useful. We recommend that a diet high in antioxidant rich foods should be developed as a habit from an early age. However, when initial signs of vision health deterioration are observed, the appropriate nutritional supplement products may be recommended but only to augment the primary medical treatments.

Allosteric inhibitors of plasma membrane Ca pumps: Invention and applications of caloxins.

Plasma membrane Ca(2+) pumps (PMCA) play a major role in Ca(2+) homeostasis and signaling by extruding cellular Ca(2+) with high affinity. PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states. Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology. We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area. A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides, caloxins may become clinically useful in cardiovascular diseases, neurological disorders, retinopathy, cancer and contraception.

Caloxin 1b3: a novel plasma membrane Ca(2+)-pump isoform 1 selective inhibitor that increases cytosolic Ca(2+) in endothelial cells.

The purpose of this study was to invent an extracellular inhibitor selective for the plasma membrane Ca(2+) pump(s) (PMCA) isoform 1. PMCA extrude Ca(2+) from cells during signalling and homeostasis. PMCA isoforms are encoded by 4 genes (PMCA1-4). Pig coronary artery endothelium and smooth muscle express the genes PMCA1 and 4. We showed that the endothelial cells contained mostly PMCA1 protein while smooth muscle cells had mostly PMCA4. A random peptide phage display library was screened for binding to synthetic extracellular domain 1 of PMCA1. The selected phage population was screened further by affinity chromatography using PMCA from rabbit duodenal mucosa which expressed mostly PMCA1. The peptide displayed by the selected phage was termed caloxin 1b3. Caloxin 1b3 inhibited PMCA Ca(2+)-Mg(2+)-ATPase in the rabbit duodenal mucosa (PMCA1) with a greater affinity (inhibition constant=17±2 µM) than the PMCA in the human erythrocyte ghosts (PMCA4, inhibition constant=45±4 µM). The affinity of caloxin 1b3 was also higher for PMCA1 than for PMCA2 and 3 indicating its selectivity for PMCA1. Consistent with an inhibition of PMCA1, caloxin 1b3 addition to the medium increased cytosolic Ca(2+) concentration in endothelial cells. Caloxin 1b3 is the first known PMCA1 selective inhibitor. We anticipate caloxin 1b3 to aid in understanding PMCA physiology in endothelium and other tissues.

Phage display: concept, innovations, applications and future.

Phage display is the technology that allows expression of exogenous (poly)peptides on the surface of phage particles. The concept is simple in principle: a library of phage particles expressing a wide diversity of peptides is used to select those that bind the desired target. The filamentous phage M13 is the most commonly used vector to create random peptide display libraries. Several methods including recombinant techniques have been developed to increase the diversity of the library. On the other extreme, libraries with various biases can be created for specific purposes. For instance, when the sequence of the peptide that binds the target is known, its affinity and selectivity can be increased by screening libraries created with limited mutagenesis of the peptide. Phage libraries are screened for binding to synthetic or native targets. The initial screening of library by basic biopanning has been extended to column chromatography including negative screening and competition between selected phage clones to identify high affinity ligands with greater target specificity. The rapid isolation of specific ligands by phage display is advantageous in many applications including selection of inhibitors for the active and allosteric sites of the enzymes, receptor agonists and antagonists, and G-protein binding modulatory peptides. Phage display has been used in epitope mapping and analysis of protein-protein interactions. The specific ligands isolated from phage libraries can be used in therapeutic target validation, drug design and vaccine development. Phage display can also be used in conjunction with other methods. The past innovations and those to come promise a bright future for this field.

Functional effects of caloxin 1c2, a novel engineered selective inhibitor of plasma membrane Ca(2+)-pump isoform 4, on coronary artery.

Coronary artery smooth muscle expresses the plasma membrane Ca(2+) pump (PMCA) isoforms PMCA4 and PMCA1. We previously reported the peptide inhibitor caloxin 1b1 that was obtained by using extracellular domain 1 of PMCA4 as the target (Am J Physiol Cell.290 [2006] C1341). To engineer inhibitors with greater affinity and isoform selectivity, we have now created a phage display library of caloxin 1b1-like peptides. We screened this library by affinity chromatography with PMCA from erythrocyte ghosts that contain mainly PMCA4 to obtain caloxin 1c2. Key properties of caloxin 1c2 are (a) Ki = 2.3 +/- 0.3 microM which corresponds to a 20x higher affinity for PMCA4 than that of caloxin 1b1 and (b) it is selective for PMCA4 since it has greater than 10-fold affinity for PMCA4 than for PMCA1, 2 or 3. It had the following functional effects on coronary artery smooth muscle: (a) it increased basal tone of the de-endothelialized arteries; the increase being similar at 10, 20 or 50 microM, and (b) it enhanced the increase in the force of contraction at 0.05 but not at 1.6 mM extracellular Ca(2+) when Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and the sarco/endoplasmic reticulum Ca(2+) pump were inhibited. We conclude that PMCA4 is pivotal to Ca(2+) extrusion in coronary artery smooth muscle. We anticipate caloxin 1c2 to aid in understanding the role of PMCA4 in signal transduction and home-ostasis due to its isoform selectivity and ability to act when added extracellularly.

Caloxins: a novel class of selective plasma membrane Ca2+ pump inhibitors obtained using biotechnology.

Plasma membrane Ca2+ pumps (PMCA) extrude cellular Ca2+ with a high affinity and hence play a major role in Ca2+ homeostasis and signaling. Caloxins (selective extracellular PMCA inhibitors) would aid in elucidating the physiology of PMCA. PMCA proteins have five extracellular domains (exdoms). Our hypotheses are: 1) peptides that bind selectively to each exdom can be invented by screening a random peptide library, and 2) a peptide can modulate PMCA activity by binding to one of the exdoms. The first caloxin 2a1, selected for binding exdom 2 was selective for PMCA (Ki=529 microM). It has been used to examine the physiological role of PMCA. PMCA isoforms are encoded by four genes. PMCA isoform expression differs in various cell types, with PMCA1 and 4 being the most widely distributed. There are differences between PMCA1-4 exdom 1 sequences, which may be exploited for inventing isoform selective caloxins. Using exdom 1 of PMCA4 as a target, modified screening procedures and mutagenesis led to the high-affinity caloxin 1c2 (Ki=2.3 microM for PMCA4). It is selective for PMCA4 over PMCA1, 2, or 3. We hope that caloxins can be used to discern the roles of individual PMCA isoforms in Ca2+ homeostasis and signaling. Caloxins may also become clinically useful in cardiovascular diseases, neurological disorders, retinopathy, cancer, and contraception.

Morning glory anomaly with bilateral choroidal colobomas in a patient with Goldenhar's syndrome.

A child with Goldenhar's syndrome, bilateral choroidal colobomas, and a morning glory anomaly of the optic disk in one eye is described. Bilateral posterior segment anomalies associated with Goldenhar's syndrome are rare. An association between the morning glory anomaly and Goldenhar's syndrome has not been previously reported.

Ca2+-pumps and Na2+-Ca2+-exchangers in coronary artery endothelium versus smooth muscle.

Vascular endothelial cells (EC) and smooth muscle cells (SMC) require a decrease in cytoplasmic Ca2+ concentration after activation. This can be achieved by Ca2+ sequestration by the sarco-/endoplasmic reticulum Ca2+ pumps (SERCA) and Ca2+ extrusion by plasma membrane Ca2+ pumps (PMCA) and Na+-Ca2+-exchangers (NCX). Since the two cell types differ in their structure and function, we compared the activities of PMCA, NCX and SERCA in pig coronary artery EC and SMC, the types of isoforms expressed using RT-PCR, and their protein abundance using Western blots. The activity of NCX is higher in EC than in SMC but those of PMCA and SERCA is lower. Consistently, the protein abundance for NCX protein is higher in EC than in SMC and those of PMCA and SERCA is lower. Based on RT-PCR experiments, the types of RNA present are as follows: EC for PMCA1 while SMC for PMCA4 and PMCA1; EC for SERCA2 and SERCA3 and SMC for SERCA2. Both EC and SMC express NCX1 (mainly NCX1.3). PMCA, SERCA and NCX differ in their affinities for Ca2+ and regulation. Based on these observations and the literature, we conclude that the tightly regulated Ca2+ removal systems in SMC are consistent with the cyclical control of contractility of the filaments and those in EC are consistent with Ca2+ regulation of the endothelial nitric oxide synthase near the cell surface. The differences between EC and SMC should be considered in therapeutic interventions of cardiovascular diseases.

Characterization of SERCA2b Ca2+-Mg2+ ATPase mRNA decay by nuclear proteins.

Gene expression is controlled at several levels including mRNA decay. Sarco/endoplasmic reticulum Ca2+-Mg2+-ATPase isoform 2b (SERCA2b) is central to Ca2+ signalling and homeostasis in several tissues. SERCA2b mRNA decay involves interactions between cis-acting elements in its 3'-region and trans-acting nuclear protein factors. In the presence of the protein factors, the synthetic capped and polyadenylated RNA fragment 2b1 (3444-3753) decays faster than other SERCA2b 3'-region fragments. Here we determined the minimum cis-acting destabilizing element in the decay and its interactions with the nuclear protein factors. The in vitro decay required ATP hydrolysis and Mg2+ but not Ca2+. The decay was directional from 3' to 5', and involved a novel 35b GC rich domain designated 2b1-4 corresponding to 3521-3555. The decay of 2b1 RNA was decreased by (a) competition with 2b1-4, (b) mutation of 2b1 to delete 2b1-4, and (c) depleting the extracts of destabilizing trans-acting factors using immobilized 2b1-4. To determine the minimal destabilizing elements 2b1-4 was divided into 7b domains A-E. Deleting AB, BC, CD or DE inactivated the destabilizing cis-acting element but deleting A, B, C, D or E had no effect. In electrophoresis mobility shift assays the nuclear protein extracts retarded the mobility of labeled uncapped 2b1 RNA without a poly A+ tail. A positive co-operativity in the interactions was shown in protein concentration dependence of the shift and in the competition of 2b1-4 in inhibiting the mobility of 2b1 RNA. Based on further experiments, the domain CDE (3535-3555) was sufficient to compete with 2b1 RNA for the protein binding. Consistent with this competition, excess CDE RNA retarded the in vitro decay of 2b1 RNA. Thus the RNA decay required ATP hydrolysis and Mg2+ but not Ca2+, the minimum binding domain was in the sequence 3535-3555, and the decay may involve a multimeric protein complex.

Hypotonic shock stimulates ascorbate release from coronary artery endothelial cells by a Ca2+ -independent pathway.

In endothelial cells, anion channels open upon osmotic swelling during shear stress and hypotonic shock. Therefore, we examined the effects of hypotonic shock on release of the antioxidant anion ascorbate from pig coronary artery endothelial cells. Hypotonic shock potentiated ascorbate release from freshly isolated or cultured pig coronary artery endothelial cells; subsequently cultured endothelial cells were used. The hypotonic shock-induced increase in Asc release was rapid, depended on the degree of hypotonic shock, and not due to membrane leakiness. Stimulating P2Y2 like receptors in endothelial cells with ATP causes ascorbate release via a Ca2+ -mediated pathway. Hypotonic shock-induced release differed from the Ca2+-mediated Asc release because: (a) the increase in release with hypotonic shock was additive to that with ATP or A23187 (Ca2+ -ionophore), (b) apyrase, suramin or removing extracellular Ca2+ did not affect the hypotonic shock-stimulated release, (c) anion channel blockers inhibited the release by the two pathways differently, and (d) hypotonic shock increased the ascorbate release from endothelial cells and cultured smooth muscle cells whereas the Ca2+ -mediated ascorbate release occurred only in endothelial cells. Accumulation of ascorbate by endothelial cells was examined at extracellular ascorbate concentrations of 10 (Na+ -ascorbate symporter not saturated) and 5000 microM (Na+ -ascorbate symporter saturated). Hypotonic shock and A23187 decreased ascorbate accumulation at 10 microM ascorbate but increased it at 5000 microM. The effects of the two treatments were additive and also differed from each other with substitution of gluconate for extracellular chloride. Thus, ascorbate release from endothelial cells can be potentiated by two distinct pathways - hypotonic shock mediated and ATP/Ca2+ stimulated.

Control of protein expression through mRNA stability in calcium signalling.

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1.

Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.

Ca2+-mediated ascorbate release from coronary artery endothelial cells.

1.--The addition of Ca(2+) ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca(2+)-mediated release. An increase in Ca(2+)-mediated Asc release was observed from PCEC preincubated with Asc, Asc-2-phosphate or dehydroascorbic acid (DHAA). 2.--The effects of various ATP analogs and inhibition by suramin were consistent with the ATP-induced release being mediated by P2Y2-like receptors. 3.--ATP-stimulated Asc release was Ca(2+)-mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca(2+)], (b) Ca(2+) ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca(2+) and chelating intracellular Ca(2+)inhibited the ATP-induced release, and (d) inositol-selective phospholipase C inhibitor U73122 also inhibited this release. 4.--Accumulation of Asc by PCEC was examined at Asc concentrations of 10 microM (Na(+)-Asc symporter not saturated) and 5 mM (Na(+)-Asc symporter saturated). At 10 microM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. 5.--PCEC but not pig coronary artery smooth muscle cells show a Ca(2+)- mediated Asc release pathway that may be activated by agents such as ATP.

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors.

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.

A novel plasma membrane Ca(2+)-pump inhibitor: caloxin 1A1.

The plasma membrane Ca(2+)-Mg(2+)-ATPase is a Ca(2+)-pump that expels Ca2+ from cells. Here we report caloxin 1A1-a novel peptide inhibitor (Ki=100 microM) of plasma membrane Ca(2+)-pump-obtained by screening a cysteine bridge-constrained random peptide library for binding to the first extracellular domain of plasma membrane Ca(2+)-pump. Dithiothreitol removed the inhibition indicating that the constraint imposed by the cysteine bridge is required for the inhibition. Caloxin 1A1 also inhibited the fast twitch sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase although weakly. Glutathione dimers (containing a cysteine bridge) inhibited the Ca(2+)-Mg(2+)-ATPase activity of sarcoplasmic reticulum Ca(2+)-Mg(2+)-ATPase, but not that of plasma membrane Ca(2+)-pump. Caloxin 1A1 stabilised Ca(2+)-dependent formation of the acid stable 140-kDa acylphosphate which is a partial reaction of this enzyme. Thus caloxin 1A1 inhibits the plasma membrane Ca(2+)-pump by perturbing the first extracellular domain indicating that the transmembrane domains 1 and 2 play a role in its reaction cycle. This finding is consistent with rearrangements that occur in transmembrane helices 1 and 2 during reaction cycle of sarcoplasmic reticulum Ca(2+)-pump. Caloxin 1A1 caused an increase in cytosolic Ca2+ concentration in endothelial cells.

Role of third extracellular domain of plasma membrane Ca2+-Mg2+-ATPase based on the novel inhibitor caloxin 3A1.

The plasma membrane Ca2+ pump (PMCA) is a Ca2+-Mg2+-ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+ ([Ca2+]i). It contains five putative extracellular domains (PEDs). Earlier we had reported that binding to PED2 leads to PMCA inhibition. Mutagenesis of residues in transmembrane domain 6 leads to loss of PMCA activity. PED3 connects transmembrane domains 5 and 6. PED3 is only five amino acid residues long. By screening a phage display library, we obtained a peptide sequence that binds this target. After examining a number of peptides related to this original sequence, we selected one that inhibits the PMCA pump (caloxin 3A1). Caloxin 3A1 inhibits PMCA but not the sarcoplasmic reticulum Ca2+-pump. Caloxin 3A1 did not inhibit formation of the 140 kDa acylphosphate intermediate from ATP or its degradation. Thus, PEDs play a role in the reaction cycle of PMCA even though sites for binding to the substrates Ca2+ and Mg-ATP2-, and the activator calmodulin are all in the cytosolic domains of PMCA. In endothelial cells exposed to low concentration of a Ca2+-ionophore, caloxin 3A1 caused a further increase in [Ca2+]i proving its ability to inhibit PMCA pump extracellularly. Thus, even though PED3 is the shortest PED, it plays key role in the PMCA function.