Patient engagement study to identify and improve surgical experience.

BACKGROUND: Patient engagement is the establishment of active partnerships between patients, families, and health professionals to improve healthcare delivery. The objective of this project was to conduct a series of patient engagement workshops to identify areas to improve the surgical experience and develop strategies to address areas identified as high priority. METHODS: Faculty surgeons and patients were invited to participate in three in-person meetings. Evaluation included identifying and developing strategies for three priority areas to improve the surgical experience and level of engagement achieved at each meeting. RESULTS: Sixteen faculty surgeons and 32 patients participated. Some 63 themes to improve the surgical experience were identified; the three highest-priority themes were physician communication, discharge process, and expectations at home after discharge. Individual improvement strategies for these three prioritized themes (12, 36 and 6 respectively) were used to develop a formal strategic plan, and included a physician communication survey, discharge process worksheet and video, and guideline regarding what to expect at home after discharge. Overall, the level of engagement achieved was considered high by over 85 per cent of the participants. CONCLUSION: A high level of patient engagement was achieved. Priorities were identified with patients and surgeons to improve surgical experience, and strategies were developed to address these areas.

Does the presence of an intact primary increase the risk of nonelective colorectal surgery in patients treated with bevacizumab?

AIM: In patients with incurable metastatic colorectal cancer (mCRC), resection of the primary tumour is debated; however, patients with intact primaries may be at a higher risk of complications requiring surgery when receiving treatment with bevacizumab. Our aim was to estimate the risk of nonelective colorectal surgery in patients undergoing bevacizumab therapy for mCRC and evaluate the association between intact primary tumours and risk of nonelective surgery. METHOD: We designed a population-based, retrospective cohort study using administrative and cancer registry data in Ontario, Canada. We included patients with mCRC who received bevacizumab from 1 January 2008 to 31 December 2014. The primary outcome was nonelective colorectal surgery after initiation of bevacizumab. We determined the cumulative incidence of nonelective colorectal surgery among patients with previously resected and unresected primaries, accounting for the competing risk of death. We explored the relationship between previous resection of the primary and need for nonelective surgery using a cause-specific hazards model, controlling for patient, tumour and treatment factors. RESULTS: We identified 1840 (32.7%) patients with intact primaries and 3784 (67.3%) patients with prior resection. The cumulative incidence of nonelective surgery 1 year after initiating bevacizumab for all patients was 3.9% (95% CI 3.4-4.5%). One-year cumulative incidence was higher in those with intact primaries than in those with resected primaries (6.1% vs 2.9%, P < 0.0001). After adjustment, an intact primary remained strongly associated with nonelective colorectal surgery (hazard ratio = 2.89, 95% CI 2.32-3.61; P < 0.0001). CONCLUSION: Bevacizumab is associated with a low but meaningful risk for serious gastrointestinal complications, necessitating vigilance, particularly among patients with an intact primary tumour.

Meta-analysis of the effect of perioperative intravenous lidocaine on return of gastrointestinal function after colorectal surgery.

BACKGROUND: Return of normal gastrointestinal (GI) function is a critical determinant of recovery after colorectal surgery. The aim of this meta-analysis was to evaluate whether perioperative intravenous (IV) lidocaine benefits return of gastrointestinal function after colorectal resection. METHODS: A comprehensive search of Ovid Medline, PubMed, Embase, Cochrane library, and clinicaltrials.org was performed on 1st July 2018. A manual search of reference lists was also performed. Inclusion criteria were as follows: randomized controlled trials (RCTs) of intravenous (IV) lidocaine administered perioperatively compared to placebo (0.9% saline infusion) as part of a multimodal perioperative analgesic regimen, human adults (> 16 years), and open or laparoscopic colorectal resectional surgery. EXCLUSION CRITERIA: non-colorectal surgery, non-placebo comparator, children, non-general anaesthetic, and pharmacokinetic studies. The primary endpoint was time to first bowel movement. Secondary endpoints were time to first passage of flatus, time to toleration of diet, nausea and vomiting, ileus, pain scores, opioid analgesia consumption, and length of stay. RESULTS: One hundred and ninety one studies were screened, with 9 RCTs meeting inclusion criteria (405 patients, four laparoscopic and five open surgery studies). IV lidocaine reduced time to first bowel movement compared to placebo [seven studies, 325 patients, mean weighted difference - 9.54 h, 95% CI 18.72-0.36, p = 0.04]. Ileus, pain scores, and length of stay were reduced with IV lidocaine compared with placebo. CONCLUSIONS: Perioperative IV lidocaine may improve recovery of gastrointestinal function after colorectal surgery. Large-scale effectiveness studies to measure effect size and evaluate optimum dose/duration are warranted.

Routine monitoring of liver function tests in lung cancer resections - a necessary burden?

BACKGROUND: Increased availability of routine investigations results in significant over-investigation, burdening patients with unnecessary tests as well as increasing cost. We aimed to identify the extent of monitoring of liver function tests in lung resections, and to ascertain whether any impact on clinical decision-making occurred. METHODS: Cases were identified using theatre records coded as "lobectomy/bilobectomy" in the three-month period 20 June 2017 to 20 September 2017. Electronic records were used to collect patient data. RESULTS: A total of 91 cases were included; 77 (85%) patients had 1 set of pre-operative LFTs, 12 (13%) patients had 2 sets, and 2 (2%) patients had 0 sets; 69 (76%) had normal LFTs pre-operatively; 298 sets of LFTs were measured post-operatively, with a median of 3 sets per patient; 61 (67%) patients had either normal or static LFTs post-operatively, 13 (14%) had isolated rise in GGT, 16 (17%) had derangement of ALT and AST, and 1 patient (1%) had deranged ALP. Altered clinical decision-making due to LFTs derangement was recorded in two cases (2%). CONCLUSION: Clinicians have an obligation to justify expense, and practise in a cost-effective manner. Our data suggest that the routine perioperative monitoring of LFTs in thoracic surgery does not give any clear benefit to patient care.

Can concordance between actual care received and a pathway map be measured on a population level in Ontario? A pilot study.

Background: Clinical pathways are associated with improved adherence to clinical guidelines; however, most studies have evaluated pathways for a single intervention at a single institution. The objective of the present study was to develop and evaluate a method of measuring concordance with a population-based clinical pathway map to determine if that method could be feasible for assessing overall health system performance. Methods: Patients with stage ii or iii colon cancer diagnosed in 2010 were identified, and clinical data were obtained through linkages to administrative databases. Pathway concordance was defined a priori based on receipt of key elements of the Ontario Health (Cancer Care Ontario) colorectal pathway maps. For stages ii and iii colon cancer alike, concordance was reported as the proportion of patients receiving care that followed the predefined key elements of the pathway map. Regression analysis was used to identify predictors of concordant care. Results: Our study identified 816 patients with stage ii and 800 patients with stage iii colon cancer. Of the patients with stage ii disease, 70% (n = 571) received concordant care. Of the patients with stage iii disease, results showed high concordance for all key elements except receipt of chemotherapy, leading to an overall concordance rate of 39% for that cohort. Conclusions: Our method of measuring concordance was feasible on a population-based level, but future studies to validate it and to develop more sophisticated methods to measure concordance in larger cohorts and various disease sites are necessary. Measurement of clinical pathway concordance on a population-based level has the potential to be a useful tool for assessing system performance.

Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells.

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c. The L-type Ca2+-channel blocker, SR7037, abolished the effects of glucose and KCl while attenuating the CCh response. Glucose-induced increases in [Ca2+]m, [Ca2+]c, and insulin secretion all demonstrate a pronounced initial peak followed by a sustained plateau. All three parameters are increased synergistically when glucose and CCh are combined. Finally, [Ca2+]m, [Ca2+]c, and insulin secretion also display desensitization phenomena following repeated additions of the three stimuli. The high sensitivity of [Ca2+]m to Ca2+ influx and the desensitization-resensitization effects can be explained by a model in which the mitochondria of INS-1 cells are strategically located to sense Ca2+ influx through plasma membrane Ca2+ channels. In conclusion, the correlation of [Ca2+]m and [Ca2+]c with insulin secretion may indicate a fundamental role for Ca2+ in the adaptation of oxidative metabolism to the generation of metabolic coupling factors and the energy requirements of exocytosis.

Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells.

Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells.

Mitochondrial dysfunction due to alterations in the mitochondrial genome (mtDNA) has recently attracted much attention, with the finding that mutations in the mitochondrially encoded proteins perturb cell function. Several disorders have been linked to such genetic changes, including a specific diabetic phenotype. Using ethidium bromide (EtBr) that intercalates into mtDNA, we have effectively eliminated functions under the control of mtDNA from the highly differentiated INS-1 insulin-secreting cell line. We have investigated the consequences on insulin secretion, mitochondrial enzyme activity, organelle structure, and membrane polarization in such cells (INS-1 rho0). Under these conditions, the mitochondrial membrane potential fails to hyperpolarize in response to either glucose or methylsuccinate. In agreement with this finding, the morphology of the mitochondria is altered in the presence of EtBr, sharing similarities with mitochondria in which the membrane potential has been collapsed with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). In addition, there is no effect of either nutrient secretagogue at the level of the plasma membrane potential, although the effect of the depolarizing agent KCl on membrane depolarization is completely preserved. Similarly, glucose and methylsuccinate fail to increase insulin secretion, whereas KCl is still effective. To test further the effects of mtDNA depletion on exocytosis, we permeabilized INS-1 cells with Staphylococcus aureus alpha-toxin, which forms small holes in the plasma membrane. In contrast to control cells, mitochondrial substrates were incapable of stimulating insulin secretion in mtDNA-deficient cells, emphasizing that the defect in secretion lies at the level of mitochondrial function rather than in the exocytotic process. The results indicate the paramount importance of the mitochondria in the downstream effects elicited by exposure to elevated concentrations of nutrient secretagogue.

Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.

In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K+) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca2+]c) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca2+-sensitive photoprotein aequorin. Resting mitochondrial free calcium concentration ([Ca2+]m) amounted to 0.41 +/- 0.18 microM (n = 40). Ang II induced a concentration-dependent (EC50 = 11.3 +/- 6.0 nM), biphasic rise of [Ca2+]m. After a large transient initial peak (5.13 +/- 0.89 microM, n = 28), [Ca2+]m decreased to a plateau that remained higher than basal [Ca2+]m for several minutes in the presence of the hormone. By contrast, studies in cells transfected with cytosolic aequorin indicated that the rise of [Ca2+]c triggered by Ang II was confined to 1.34 +/- 0.26 microM (n = 17). In Ca2+-free medium, a reduced peak [Ca2+]m response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca2+, in the presence of the hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca2+]m. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca2+]m rise but not the [Ca2+]c response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 mM) induced a sustained [Ca2+]c response, which was relayed without amplification into the mitochondrial matrix as a plateau of[Ca2+]m. This plateau of[Ca2+]m was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca2+]m following the peak induced by Ang II. In cells whose [Ca2+]c was clamped at various levels (0.05-0.860 microM) with ionomycin, a concentration-dependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone--the early product of steroidogenesis--was markedly potentiated by CGP37157. These results suggest the existence of microdomains of high [Ca2+]c elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade.

Role of mitochondrial calcium in metabolism-secretion coupling in nutrient-stimulated insulin release.

Glucose-stimulated insulin release from pancreatic beta cells involves a complex series of signalling pathways. In many forms of diabetes, lesions in this process cause or aggravate the diabetic phenotype. A common motif in these cascades is the elevation of intracellular Ca2+ both in the cytosolic compartment ([Ca2+]c) and within the mitochondria ([Ca2+]m). These parameters can be effectively monitored using the photoprotein aequorin which can be targeted to subcellular compartments by transfection. It is shown that physiological concentrations of glucose elicit [Ca2+]c oscillations measured with fura-2, which correlate well with oscillatory NAD(P)H fluorescence in the mitochondria. Aequorin measurements of [Ca2+]m, though unable to detect oscillations on a single cell basis, reveal large increases in intraorganellar [Ca2+] in response to glucose, elevated amino acid levels and depolarizing concentrations of KCI. These oscillations, in turn, mirror changes in the insulin secretion profile. Since several of the key mitochondrial dehydrogenases involved in oxidative phosphorylation are exquisitely sensitive to changes in [Ca2+], it is proposed that alterations in [Ca2+]m lead to increased activity of the tricarboxylic acid cycle and subsequent ATP production, thereby facilitating exocytosis of insulin from secretory granules. The involvement of the mitochondria in these processes is examined, as is the putative role of efficient mitochondrial genome transcription and translation in normal and diabetic states.

Changes in extracellular calcium within the physiological range influence receptor-mediated inositol phosphate responses in brain and tracheal smooth muscle slices.

The effect of changes in extracellular calcium concentration ([Ca2+]e) on the incorporation of myo-[2-3H]-inositol into phosphoinositides and agonist-stimulated 3H-inositol phosphates (3H-InsPs) was examined in rat cerebral cortex and bovine tracheal smooth muscle slices. In brain slices, reduction in [Ca2+]e from 2.4 to 1.2 mmol/l resulted in an approximate doubling of the carbachol and noradrenaline-stimulated 3H-InsP response with no effect on the EC50 values. An identical effect of varying [Ca2+]e was observed for carbachol-stimulated 3H-InsP formation in tracheal smooth muscle with a further increase in 3H-InsPs evident at [Ca2+]e 0.6 mmol/l. In this tissue the effect of changes in [Ca2+]e on the incorporation of myo-[2-3H]-inositol into the total phosphoinositide pool directly paralleled the changes in 3H-InsPs except in conditions of no added calcium when 3H-InsP responses were markedly impaired. Additional studies in brain slices using buffer where the added calcium varied between 0 and 2.4 mmol/l, showed that both the carbachol stimulated formation of separate inositol phosphates during short incubation periods and incorporation of myo-[2-3H]-inositol into PtdInsP and PtdInsP2 under basal conditions was maximal at [Ca2+]e 0.3 mmol/l. Omitting Ca2+ from the buffer resulted in maximal labelling of PtdIns but a decrease in PtdInsP and PtdInsP2 labelling (compared with the level at [Ca2+]e 0.3 mmol/l) and a markedly impaired inositol polyphosphate response. Alterations in [Ca2+]e following 3H-inositol labelling but immediately prior to carbachol stimulation did not influence 3H-inositol polyphosphate responses. It is therefore clear that even relatively small changes in [Ca2+]e markedly influence agonist-stimulated 3H-InsP responses in brain and tracheal smooth muscle slices and that these reflect changes in the labelling of substrate inositol lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Can the standard gamble be used to determine utilities for uncertain health states? An example using postoperative maintenance therapy in Crohn's disease.

The objective of this study was to determine whether patients with Crohn's disease (CD) value the absolute reduction in postoperative recurrence risk attributable to therapy with mesalamine (5-ASA). One hundred subjects evaluated state A (taking 5-ASA; 25% risk of recurrence), state B (not taking 5-ASA; 40% risk of recurrence), and state C (100% risk of recurrence) by rank order, visual analog scale (VAS), and standard gamble (SG). Sixty-five of 91 patients (71%) with completed and usable questionnaires had the same preference order for state A (25% risk), state B (40% risk), and state C (100% risk) on both the VAS and the SG. The mean scores for state A (25% risk), state B (40% risk), and state C (100% risk), respectively, were 67.5, 49.8, and 19.8 on the VAS and 0.977, 0.972, and 0.910 on the SG. Subgroup analyses using stepwise logistic regression showed that risk attitude seemed to be predictive of subjects' preferences for 5-ASA. These results suggest that most subjects seem to value the 15% absolute risk reduction offered by 5-ASA. Furthermore, the SG seems to be a feasible method for measuring utilities for uncertain health states in patients with CD.

Patients' perceptions of their participation in a clinical trial for postoperative Crohn's disease.

OBJECTIVE: To explore patients' perceptions of their participation in a randomized controlled trial. PATIENTS AND METHODS: A 27-item questionnaire was mailed to all patients who participated in a randomized controlled trial that determined the effectiveness of mesalamine in preventing the recurrence of Crohn's disease postoperatively. RESULTS: The response rate was 66% (99 of 149). Fifty-five per cent of the patients felt that they received better medical care than they otherwise would have and 53% liked taking the medication. Sixty-eight per cent of the patients did not feel that annual colonoscopy was too frequent and 81% felt that the time commitment did not significantly interfere with their job or other activities. Seventy-five per cent and 62% of the patients would have liked more information and education, respectively, about Crohn's disease incorporated into the trial. Although 91% of the patients would agree to participate in a future randomized controlled trial comparing medical therapies, only 44% would agree to participate in a future randomized controlled trial comparing medical with surgical therapies. CONCLUSIONS: The majority of patients were satisfied with their participation in the trial. A large proportion of the patients would participate again but would like more information and education incorporated into the trial. Furthermore, post-trial questionnaires may be helpful in the design of future trials.

The UK Diabetes Research Network--an opportunity and a challenge.

The Department of Health has funded a national diabetes network to support clinical research. The network will facilitate recruitment into clinical trials and has been widely welcomed by clinicians. However, if the network is to reach its full potential, all those involved will need to advocate a change in attitude towards clinical trials and research, encouraging participation and contribution of data. Clinicians need to be willing to take a proactive view about research studies, and to encourage patients to adopt a positive and altruistic attitude towards trial participation. The future of trials and other important clinical research in the UK may depend on it.

Increased experience and surgical technique lead to improved outcome after ileal pouch-anal anastomosis: a population-based study.

PURPOSE: This study was designed to determine whether changes in length of stay and 30-day readmission, reoperation, and excision rates for the ileal pouch-anal anastomosis occurred over time and with changes in surgical technique and hospital volume. METHODS: Using three population-based administrative databases, data on all ileal pouch-anal anastomoses performed in the province of Ontario between January 1992 and June 1998 were obtained. The effect of age, gender, stage of the procedure, year of surgery, and hospital volume were examined for their effect on length of stay and readmission, reoperation, and excision rates. RESULTS: There were 1,285 ileal pouch-anal anastomoses performed in 58 hospitals. There was a significant decrease in length of stay and reoperation and excision rates but a concommitant increase in readmission rate during the study period. Patients younger than aged 40 years had a significantly lower length of stay and excision rate. Patients who had a two-stage procedure had a shorter length of stay, readmission, and reoperative rate compared with those having a three-stage procedure. Hospital volume was a significant predictor of need for reoperation and excision with both low-volume and medium-volume hospitals having significantly higher rates than high-volume hospitals. CONCLUSIONS: Outcome after ileal pouch-anal anastomosis has improved. It is significantly better in patients younger than aged 40 years, having a two-stage procedure, and where surgery is performed at high-volume hospitals. It is likely that both modifications in surgical technique and surgical experience have led to improvements in clinical outcome after ileal pouch-anal anastomosis.

Colon and rectal anastomoses do not require routine drainage: a systematic review and meta-analysis.

OBJECTIVE: Many surgeons continue to place a prophylactic drain in the pelvis after completion of a colorectal anastomosis, despite considerable evidence that this practice may not be useful. The authors conducted a systematic review and meta-analysis of randomized controlled trials to determine if placement of a drain after a colonic or rectal anastomosis can reduce the rate of complications. METHODS: A search of the Medline database of English-language articles published from 1987 to 1997 was conducted using the terms "colon," "rectum," "postoperative complications," "surgical anastomosis," and "drainage." A manual search was also conducted. Four randomized controlled trials, including a total of 414 patients, were identified that compared the routine use of drainage of colonic and/or rectal anastomoses to no drainage. Two reviewers assessed the trials independently. Trial quality was critically appraised using a previously published scale, and data on mortality, clinical and radiologic anastomotic leakage rate, wound infection rate, and major complication rate were extracted. RESULTS: The overall quality of the studies was poor. Use of a drain did not significantly affect the rate of any of the outcomes examined, although the power of this analysis to exclude any difference was low. Comparison of pooled results revealed an odds ratio for clinical leak of 1.5 favoring the control (no drain) group. Of the 20 observed leaks among all four studies that occurred in a patient with a drain in place, in only one case (5%) did pus or enteric content actually appear in the effluent of the existing drain. CONCLUSIONS: Any significant benefit of routine drainage of colon and rectal anastomoses in reducing the rate of anastomotic leakage or other surgical complications can be excluded with more confidence based on pooled data than by the individual trials alone. Additional well-designed randomized controlled trials would further reinforce this conclusion.

Lithium reduces the accumulation of inositol polyphosphate second messengers following cholinergic stimulation of cerebral cortex slices.

The ability of lithium to interfere with the metabolism of inositol phosphates in brain may underlie its therapeutic action in manic-depressive illness. In these experiments, lithium, at therapeutic concentrations, enhanced the accumulation of [3H]inositol monophosphate but suppressed the accumulation of the putative second messengers [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) and [3H]inositol 1,3,4,5-tetrakisphosphate following stimulation of cerebral cortex slices with carbachol. Mass measurements of Ins(1,4,5)P3 showed similar inhibitory effects, which could be prevented by preincubation with myo-inositol. These data may reveal the mechanism by which lithium can reduce polyphosphoinositide-mediated neurotransmission in brain.

Mitochondrial activation directly triggers the exocytosis of insulin in permeabilized pancreatic beta-cells.

In the pancreatic beta-cell, insulin secretion is stimulated by glucose metabolism resulting in membrane potential-dependent elevation of cytosolic Ca2+ ([Ca2+]c). This cascade involves the mitochondrial membrane potential (delta psi[m]) hyperpolarization and elevation of mitochondrial Ca2+ ([Ca2+]m) which activates the Ca(2+)-sensitive NADH-generating dehydrogenases. Metabolism-secretion coupling requires unidentified signals, other than [Ca2+]c, possibly generated by the mitochondria through the rise in [Ca2+]m. To test this paradigm, we have established an alpha-toxin permeabilized cell preparation permitting the simultaneous monitoring of [Ca2+] with mitochondrially targeted aequorin and insulin secretion under conditions of saturating [ATP] (10 mM) and of clamped [Ca2+]c at substimulatory levels (500 nM). The tricarboxylic acid (TCA) cycle intermediate succinate hyperpolarized delta psi(m), raised [Ca2+]m up to 1.5 microM and stimulated insulin secretion 20-fold, without changing [Ca2+]c. Blockade of the uniporter-mediated Ca2+ influx into the mitochondria abolished the secretory response. Moreover, glycerophosphate, which raises [Ca2+]m by hyperpolarizing delta psi(m) without supplying carbons to the TCA cycle, failed to stimulate exocytosis. Activation of the TCA cycle with citrate evoked secretion only when combined with glycerophosphate. Thus, mitochondrially driven insulin secretion at permissive [Ca2+]c requires both a substrate for the TCA cycle and a rise in [Ca2+]m. Therefore, mitochondrial metabolism generates factors distinct from Ca2+ and ATP capable of inducing insulin exocytosis.

Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells.

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+]er) is important for protein processing and signal transduction. In the pancreatic beta-cell, dysregulation of [Ca2+]er may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+]er was 267 +/- 9 microM. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+]er by 20-25%. Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+]er in the potency order K+ >> glucose > leucine, paralleling their actions in the cytosolic compartment. Glucose, which augmented [Ca2+]er by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+]er, but only the former generated whereas the latter consumed ATP. Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+]er, emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+]er, invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+]er to ensure beta-cell homeostasis.