N-acetyl-L-leucine for Niemann-Pick type C: a multinational double-blind randomized placebo-controlled crossover study.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code "IB1001-301") for the chronic treatment of symptoms in adult and pediatric patients with NPC. METHODS: This multinational double-blind randomized placebo-controlled crossover phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. DISCUSSION: Pre-clinical as well as observational and phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. TRIAL REGISTRATIONS: The trial (IB1001-301) has been registered at www. CLINICALTRIALS: gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10). Registered on 20 December 2021.

A master protocol to investigate a novel therapy acetyl-L-leucine for three ultra-rare neurodegenerative diseases: Niemann-Pick type C, the GM2 gangliosidoses, and ataxia telangiectasia.

BACKGROUND: The lack of approved treatments for the majority of rare diseases is reflective of the unique challenges of orphan drug development. Novel methodologies, including new functionally relevant endpoints, are needed to render the development process more feasible and appropriate for these rare populations and thereby expedite the approval of promising treatments to address patients' high unmet medical need. Here, we describe the development of an innovative master protocol and primary outcome assessment to investigate the modified amino acid N-acetyl-L-leucine (Sponsor Code: IB1001) in three separate, multinational, phase II trials for three ultra-rare, autosomal-recessive, neurodegenerative disorders: Niemann-Pick disease type C (NPC), GM2 gangliosidoses (Tay-Sachs and Sandhoff disease; "GM2"), and ataxia telangiectasia (A-T). METHODS/DESIGN: The innovative IB1001 master protocol and novel CI-CS primary endpoints were developed through a close collaboration between the Industry Sponsor, Key Opinion Leaders, representatives of the Patient Communities, and National Regulatory Authorities. As a result, the open-label, rater-blinded study design is considerate of the practical limitations of recruitment and retention of subjects in these ultra-orphan populations. The novel primary endpoint, the Clinical Impression of Change in Severity© (CI-CS), accommodates the heterogenous clinical presentation of NPC, GM2, and A-T: at screening, the principal investigator appoints for each patient a primary anchor test (either the 8-m walk test (8MWT) or 9-hole peg test of the dominant hand (9HPT-D)) based on his/her unique clinical symptoms. The anchor tests are videoed in a standardized manner at each visit to capture all aspects related to the patient's functional performance. The CI-CS assessment is ultimately performed by independent, blinded raters who compare videos of the primary anchor test from three periods: baseline, the end of treatment, and the end of a post-treatment washout. Blinded to the time point of each video, the raters make an objective comparison scored on a 7-point Likert scale of the change in the severity of the patient's neurological signs and symptoms from video A to video B. To investigate both the symptomatic and disease-modifying effects of treatment, N-acetyl-L-leucine is assessed during two treatment sequences: a 6-week parent study and 1-year extension phase. DISCUSSION: The novel CI-CS assessment, developed through a collaboration of all stakeholders, is advantageous in that it better ensures the primary endpoint is functionally relevant for each patient, is able to capture small but meaningful clinical changes critical to the patients' quality of life (fine-motor skills; gait), and blinds the primary outcome assessment. The results of these three trials will inform whether N-acetyl-L-leucine is an effective treatment for NPC, GM2, and A-T and can also serve as a new therapeutic paradigm for the development of future treatments for other orphan diseases. TRIAL REGISTRATION: The three trials (IB1001-201 for Niemann-Pick disease type C (NPC), IB1001-202 for GM2 gangliosidoses (Tay-Sachs and Sandhoff), IB1001-203 for ataxia telangiectasia (A-T)) have been registered at www.clinicaltrials.gov (NCT03759639; NCT03759665; NCT03759678), www.clinicaltrialsregister.eu (EudraCT: 2018-004331-71; 2018-004406-25; 2018-004407-39), and https://www.germanctr.de (DR KS-ID: DRKS00016567; DRKS00017539; DRKS00020511).

Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Opening the black box of cancer surgery quality: WebSMR and the Alberta experience.

A web-based synoptic operative report, the WebSMR (Surgical Medical Record), was developed to define and improve the quality of cancer surgery. Surgeons accurately record the essential steps of an operation including important decision-making in an analyzable format. Outcomes can be reviewed with provincial aggregates for quality improvement and maintenance of certification. Future synoptic pathology and follow-up templates will open the "black box" of surgical processes to define quality indicators for the improvement of cancer outcomes.

Increased signs of acute rejection with ischemic time in a rat musculocutaneous allotransplant model.

BACKGROUND: Composite tissue allotransplantation (CTA) may restore a variety of tissue defects, but carries the potential risks of graft failure and/or immunosuppression-related complications. Ischemia-reperfusion injury has been documented in CTA is known to contribute to acute rejection of solid organ grafts. This study describes the influence of subcritical ischemic time (ie, ischemia sufficient to generate reversible cell damage) on signs of rejection of musculocutaneous allograft components of subcritical ischemic time, namely, ischemia sufficient to generate reversible cell injury. Although skin is considered the most antigenic component of a composite allograft and is currently used for rejection surveillance, muscle and adipose are more susceptible to ischemia-related injury. METHODS: Vascularized epigastric flaps were transplanted from WKY to Fisher 344 rats after 1 or 3 hours of ischemia. Biopsies taken on postoperative day 6 were graded for signs of acute rejection according to criteria modified from previously published grading systems for CTA rejection. RESULTS: Skin and muscle exposed to 3 hours of ischemia showed significantly higher rejection scores than after 1 hour of ischemia, as evidenced by a more aggressive diffuse lymphocytic infiltration with disruption of tissue architecture. The rejection score in skin with 3-hour ischemia was 5.0 +/- 0.1 versus 3.7 +/- 0.2 with 1-hour (Mann-Whitney U test; P < .05). The rejection score in muscle exposed to 3-hour ischemia was 3.6 +/- 0.3 versus 2.5 +/- 0.1 with 1-hour (P < .05). CONCLUSIONS: Muscle and skin demonstrated increased acute rejection of allotransplants with increased subcritical ischemic time. This study supports the use of aggressive methods to reduce subcritical ischemic injury during allotransplantation of composite tissue and inclusion of muscle in postoperative biopsies in this early investigational period of CTA.

Utilizing patient satisfaction surveys to prepare for Medicaid managed care.

To prepare for Medicaid managed care, a community health center incorporated the business principle of continuous quality improvement, often used in the private sector to improve customer service, into its planning process. The initial endeavor was to create a patient satisfaction survey that was appropriate for the uniqueness of the community. The survey, taken monthly, resulted in both staff and patients making active improvements in the clinic environment. Staff showed more enthusiasm, and patients were more assertive in their attitudes toward the clinic. The empowerment of the patient to take ownership in the clinic will be coupled with the next step of the formalized plan, that of educating patients on the steps necessary to ensure that their Medicaid managed care facility will be the local community health center.

Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release.

The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis. In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12. Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins. Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins. Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation. These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation.

Gi/Go couple met-enkephalin to inhibition of cholinergic and beta-adrenergic stimulation of lacrimal secretion.

PURPOSE: To determine whether G-protein-mediated inhibition of secretion by met-enkephalin involves cyclic adenosine monophosphate (cAMP)-dependent events and to identify the G proteins that couple met-enkephalin to inhibition of lacrimal secretion. METHODS: Secretion of protein was measured in 3-day primary cultures of rabbit lacrimal acini exposed to vehicle, the cholinergic agonist carbachol (Cch), the beta-adrenergic agonist isoproterenol (Isop), vasoactive intestinal peptide (VIP), or forskolin (FSK) with or without the enkephalin analog D-ala2-met-enkephalinamide (DALA). In separate experiments, cells were pretreated with pertussis toxin or polyclonal antibodies against the alpha subunits of Gi/Go to determine the physiologic role of G proteins in met-enkephalin inhibition of the release of lacrimal protein. Adenylyl cyclase (AC) activity was measured by a cAMP-dependent protein kinase binding assay in lacrimal membranes in response to the same agonists used in the secretion studies. RESULTS: Cch resulted in a significant increase in protein release from cultured lacrimal acini. Increased secretion also occurred with Isop, VIP, and FSK. Cch- and Isop-stimulated secretion was inhibited by DALA to near-basal values. However, DALA did not inhibit VIP- or FSK-stimulated secretion. The inhibitory effect of DALA on Cch and Isop stimulation of secretion was reversed by pertussis toxin. Inhibition of Cch-stimulated secretion was blocked by antibody specific to a common peptide sequence of Gialpha1 and Gialpha2 but was not blocked by anti-Gialpha1 antibody. The inhibitory effect on Cch-stimulated secretion was also blocked by anti-Gialpha3 and anti-Goalpha. Similar experiments resulted in a reversal of DALA inhibition of beta-adrenergic stimulation of secretion by immunoneutralization of Gialpha1/2 and Goalpha but not by immunoneutralization of Gialpha1 or Gialpha3. VIP, Isop, and FSK significantly stimulated AC. However, Cch had no effect on the activity of the enzyme. In addition, DALA had no effect on AC activity under any conditions. CONCLUSIONS: These results show that enkephalin inhibition of cholinergic and beta-adrenergic stimulation of secretion is mediated by Gi2, Gi3, and Go. The effector coupled by the G proteins is not AC. However, we suggest a role for met-enkephalin in G-protein-coupled modulation of ion channels important for cholinergic and beta-adrenergic stimulation of lacrimal secretion.

Adrenergic stimulation of lacrimal protein secretion is mediated by G(q/11)alpha and G(s)alpha.

PURPOSE: The intent of this work was to continue the characterization of G protein coupling of receptors to lacrimal secretion by determination of whether the alpha subunits of the heterotrimeric G proteins G(q/11) and G(s) couple alpha and beta-adrenergic receptor activation to stimulation of protein secretion by isolated lacrimal acini. In addition, we assessed the possibility that the G beta gamma dimer influences stimulated lacrimal protein secretion. METHODS: Primary cultures of rabbit lacrimal acini were permeabilized by streptolysin-O (SLO) to allow cellular insertion of polyclonal antibodies to G(q/ll)alpha, G(s)alpha or G beta or GDP beta S. Following this, secretion of exocytotic protein was measured in response to vehicle, the alpha(1)-adrenergic agonist phenylephrine, the beta-adrenergic agonist isoproterenol, the cholinergic agonist carbachol or vasoactive intestinal peptide (VIP). RESULTS: Phenylepherine and isoproterenol resulted in a significant increase in protein release from cultured lacrimal acini. The increase in secretion elicited by the adrenergic agonists, however, was much less than that induced by either carbachol or VIP. Antibody to G(q/11)alpha blocked phenylephrine stimulated secretion 32% but had no effect on isoproterenol or VIP stimulation of secretion. In contrast, the same antibody blocked carbachol stimulated secretion by 72%. Antibody to G(s)alpha blocked isoproterenol stimulated secretion 20%, had no effect on phenylephrine stimulation, blocked carbachol stimulation by 27% and VIP stimulation by 69%. Antibody to G beta did not effect stimulation of secretion by any agonist. The degree of inhibition of secretion following exposure to GDPbetaS did not exceed that obtained with the G protein subunit antibodies. CONCLUSIONS: The alpha subunit of G(q/11) couples activation of alpha(1)-adrenergic receptors to exocytotic release of protein in the lacrimal gland. Activation of beta-adrenergic receptors does not involve G(q/11) but is mediated by the alpha subunit of G(s). G protein coupling of the adrenergic receptors to secretion appears to be limited compared to cholinergic and VIP stimulation and might suggest the occurrence of the activation of intracellular signaling pathways independent of receptor-G protein-effector regulation of adrenergic stimulation of lacrimal secretion.

Cerebral microdialysis combined with single-neuron and electroencephalographic recording in neurosurgical patients. Technical note.

Monitoring physiological changes in the brain parenchyma has important applications in the care of neurosurgical patients. A technique is described for measuring extracellular neurochemicals by cerebral microdialysis with simultaneous recording of electroencephalographic (EEG) and single-unit (neuron) activity in selected targets in the human brain. Forty-two patients with medically intractable epilepsy underwent stereotactically guided implantation of a total of 423 intracranial depth electrodes to delineate potentially resectable seizure foci. The electrodes had platinum alloy contacts for EEG recordings and four to nine 40-microm microwires for recording single-unit neuron activity. Eighty-six electrodes also included microdialysis probes introduced via the electrode lumens. During monitoring on the neurosurgical ward, electrophysiological recording and cerebral microdialysis sampling were performed during seizures, cognitive tasks, and sleep-waking cycles. The technique described here could be used in developing novel approaches for evaluation and treatment in a variety of neurological conditions such as head injury, subarachnoid hemorrhage, epilepsy, and movement disorders.

Executive directors and community health centers--facing the healthcare transition.

This paper examines the impressions and experiences of administrators who manage Community Health Centers (CHCs) in Region VI, US Department of Health and Human Services, with the goal of identifying leadership skills and intrinsic values that are needed to run integrated service delivery sites. As the delivery of healthcare service shifts to health promotion and disease prevention, Community Health Centers are well positioned to assume major roles in this transition. However, some CHC administrators may need additional skills in order to address the changing healthcare environment. A survey of CHC Executive Directors was conducted to identify their impressions and experiences. Information obtained from this exploratory study should be beneficial in educating the next generation of healthcare administrators.

Gi2 and Gi3 couple met-enkephalin to inhibition of lacrimal secretion.

PURPOSE: The intent of this study was to identify the pertussis toxin-sensitive G proteins that couple met-enkephalin to the inhibition of cholinergically stimulated secretion in rabbit lacrimal gland acini. METHODS: The authors detected G proteins in membranes from freshly isolated glands, freshly isolated acini, and cultured lacrimal acini from rabbits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Antibodies against the alpha subunits of Gi1, Gi1 and Gi2, or Gi3 were used in cultured acini permeabilized by streptolysin-O to determine the role of the G proteins in met-enkephalin inhibition of cholinergic stimulation of lacrimal acinar protein release. RESULTS: Western blot analysis showed the presence of the alpha subunits of Gi2 and Gi3, but not Gi1, in all three membrane preparations. The met-enkephalin analog D-Ala2-methionine enkephalinamide (DALA) inhibited cholinergic stimulation of secretion by cultured rabbit acinar cells to near basal levels. Inhibition of secretion by DALA was blocked by insertion of antibody to a peptide sequence common to Gialpha1 and Gialpha2, but was not blocked by antibody against a specific Gialpha1 sequence. The inhibitory effect of DALA also was blocked by antibody to a Gialpha3 sequence. At low doses of anti-Gialpha1/2 and anti-Gialpha3 in combination, the effect on reversal of inhibition was additive. However, at higher doses, the effect of the combination was no greater than the effect of either antibody alone. CONCLUSIONS: These results demonstrate that met-enkephalin inhibition of cholinergic secretion is mediated by way of the pertussis toxin-sensitive G proteins Gi2 and Gi3 in cultured rabbit lacrimal acini. Because the effects of the G proteins are not additive, the intracellular events distal to G protein activation most likely converge at some point before exocytosis.

Accuracy of fluorocrit in determination of blood perflubron concentration.

Previous studies examining the radiosensitizing effects of perfluorochemical emulsions have based dose recommendations on a measurement known as fluorocrit. The fluorocrit is the proportion of blood volume occupied by perfluorochemicals and is measured using standard hematocrit procedures. This measurement is inherently crude and subject to error and variability between different individuals measuring the same sample. Furthermore, the fluorocrit method has not been compared to other quantitative methods to determine its reliability. The purpose of this study was to compare fluorocrit measurements to those obtained by gas chromatographic analysis. A 90% w/v perflubron emulsion was administered to six normal dogs once weekly for four weeks and peripheral blood samples were obtained at specified time points for analysis. A total of 123 blood samples were analyzed by both methods. The relationship between blood fluorocrit and plasma perflubron concentration measured by gas chromatography was examined using regression models. Based on the modest predictive value (r2 = 0.3683) of the derived statistical model, we conclude that fluorocrit measurement is an inaccurate method of estimation of blood perflubron concentration. Caution must, therefore, be exercised when extrapolating data and dose recommendations from reports of studies using flurocrit as the only estimate of blood perflubron concentration.

Identification of a GTPase activating protein specific for the heterotrimeric G protein, Gz.

Gz is a member of the Gi family of trimeric G proteins whose precise signalling function has not been defined. It can be distinguished from other members of the family by several interesting biochemical properties of its alpha subunit, Gzalpha. One particularly intriguing property is its extremely slow GTPase activity; its kcat for GTP hydrolysis is as much as 200-fold less than other Galpha's. Since there is evidence that cellular factors can accelerate the GTPase activities of Galpha subunits, we have suspected that cells expressing Gzalpha may contain a GTPase-activating protein, or GAP, that would enhance its hydrolytic ability. Using purified Gzalpha-GTP as a substrate, we have identified and characterized such a GAP that acts on Gzalpha, which we have termed Gz-GAP. The protein responsible for this activity is specific for Gzalpha and is found in the membrane fraction of bovine brain and in several other tissues that express Gz. Since G protein effectors are in many cases capable of stimulating the GTPases rate of Galpha subunits, we speculate that a novel effector for Gz is responsible for the activity.

Lymphocyte recruitment and the kinetics of adhesion receptor expression during the pulmonary immune response to particulate antigen.

The selectins and beta2 integrins participate in the recruitment of neutrophils in acute pulmonary inflammation. However, the cell adhesion receptors that mediate lymphocyte trafficking into the lung have not been defined. This study examined the relationship between cell adhesion molecules on the pulmonary vasculature and on lymphocytes recovered from the lung during a pulmonary immune response to intratracheal (I.T.) sheep red blood cells (SRBCs) in sensitized C57BL/6J mice. Silver-enhanced immunogold staining and reverse transcriptase polymerase chain reaction of lung tissues revealed sustained induction of VCAM-1, E-selectin, and P-selectin on the pulmonary vasculature for up to 7 days after I.T.-SRBC challenge. Neither the MECA 79 nor MECA 367 antigens were induced on the pulmonary vasculature during this period. In the peripheral blood, both CD4 and CD8 T-cell subsets showed an initial increase in P-selectin ligand expression after I.T.-SRBC challenge. The number of P-selectin ligand-positive T cells in the peripheral blood fell as T cells with both P-selectin and, to a lesser extent, E-selectin ligands accumulated in the bronchoalveolar lavage fluid. We conclude that I.T.-SRBC challenge in sensitized mice elicits prolonged synthesis of P-selectin, E-selectin, and VCAM-1 by the lung vasculature as well as selectin ligand synthesis by responding T cells. Furthermore, the entry of selectin-ligand-positive T cells into the circulation and their accumulation in the bronchoalveolar lavage fluid indicates that these receptors may contribute to T cell recruitment. Finally, VCAM-1 on the vasculature may also participate; however, the vascular addressins, required for homing to peripheral and mucosal lymphoid organs, are not essential for T-cell entry into the lung following I.T.-SRBC challenge.

Gs and Gq/11 couple vasoactive intestinal peptide and cholinergic stimulation to lacrimal secretion.

PURPOSE: The intent of this study was to determine the physiological role of selected G proteins in receptor-mediated protein release by lacrimal acini. METHODS: The role of G proteins in lacrimal secretion was determined in tissues obtained from the lacrimal glands of adult male New Zealand White rabbits. Pertussis toxin treatment of primary acinar cultures and permeabilization of cultured acini with streptolysin-O and insertion of GDP beta S or antibodies against the alpha subunit of Gs or Gq/11 were used to determine the role of G proteins in vasoactive intestinal peptide (VIP) and carbachol-stimulated lacrimal secretion. Gs and Gq/11 were identified in lacrimal membranes obtained from freshly isolated lacrimal gland fragments, freshly isolated acini, and cultured acini by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. RESULTS: Permeabilization by streptolysin-O and introduction of guanosine thiodiphosphate into cultured acini blocked stimulation of protein released by either 100 nM VIP or 100 microM carbachol by approximately 50%. Exposure of cultured acini to 100 ng/ml pertussis toxin for 36 to 48 hours did not affect stimulated release by either agonist, indicating that the guanosine triphosphate-dependent actions of VIP and carbachol are mediated through pertussis toxin-insensitive G proteins. Pertussis toxin-insensitive G proteins in lacrimal membranes obtained from freshly isolated glands, freshly isolated acini, and cultured acini were identified with polyclonal antibodies to the alpha subunits of Gs and Gq/11. Immunoblotting of lacrimal membranes with anti-Gs alpha antiserum showed two immunoreactive bands at 44 and 47 kDa. Anti-Gq/11 alpha antiserum detected a single band at 46 kDa in similar membrane preparations. Anti-Gs alpha antiserum reduced the secretory response to VIP by 64% and to carbachol by 37%. Introduction of anti-Gq/11 alpha antiserum reduced the response to carbachol by 70%; however, the response to VIP was unchanged. Simultaneous introduction of both antisera caused no further reduction of VIP-stimulated release than did anti-Gs alpha antiserum alone. However, simultaneous introduction of both anti-Gs alpha and anti-Gq/11 alpha antisera resulted in complete inhibition of the effects of carbachol on protein release by cultured acini. CONCLUSIONS: These results show that VIP receptor activation of lacrimal protein release is mediated through Gs, whereas cholinergic stimulation involves both Gs and Gq/11. From the authors' results, the authors conclude that Gs links VIP receptor activation to adenylyl cyclase and cyclic adenosine 3'-5' monophosphate production and the ultimate release of protein by acinar cells and that Gq/11 links muscarinic receptor activation to phospholipase C and IP3 and diacylglycerol accumulation, which also leads to protein release. Furthermore, it is hypothesized that Gs has an additional role in the regulation of vesicular traffic and exocytosis.

Signalling functions and biochemical properties of pertussis toxin-resistant G-proteins.

Pertussis toxin (PTX) has been widely used as a reagent to characterize the involvement of heterotrimeric G-proteins in signalling. This toxin catalyses the ADP-ribosylation of specific G-protein alpha subunits of the Gi family, and this modification prevents the occurrence of the receptor-G-protein interaction. This review focuses on the biochemical properties and signalling of those G-proteins historically classified as 'PTX-resistant' due to the inability of the toxin to influence signalling through them. These G-proteins include members of the Gq and G12 families and one Gi family member, i.e. Gz. Signalling pathways controlled by these G-proteins are well characterized only for Gq family members, which activate specific isoforms of phospholipase C, resulting in increases in intracellular calcium and activation of protein kinase C (PKC), among other responses. While members of the G12 family have been implicated in processes that regulate cell growth, and Gz has been shown to inhibit adenylate cyclase, the specific downstream targets to these G-proteins in vivo have not been clearly established. Since two of these proteins, G12 alpha and Gz alpha, are excellent substrates for PKC, there is the potential for cross-talk between their signalling and Gq-dependent processes leading to activation of PKC. In tissues that express these G-proteins, a number of guanine-nucleotide-dependent, PTX-resistant, signalling pathways have been defined for which the G-protein involved has not been identified. This review summarizes these pathways and discusses the evidence both for the participation of specific PTX-resistant G-proteins in them and for the regulation of these processes by PKC.