Endothelial nitric-oxide synthase (eNOS), a Ca2+/calmodulin-dependent enzyme, is critical for vascular homeostasis. While eNOS is membrane-associated through itsN-myristoylation, the significance of ...membrane association in locating eNOS near sources of Ca2+ entry is uncertain. To assess the Ca2+ source required for eNOS activation, chimera containing the full-length eNOS cDNA and HA-tagged aequorin sequence (EHA), and MHA (myristoylation-deficient EHA) were generated and transfected into COS-7 cells. The EHA chimera was primarily targeted to the plasma membrane while MHA was located intracellularly. Both constructs retained enzymatic eNOS activity and aequorin-mediated Ca2+ sensitivity. The plasma membrane-associated EHA and intracellular MHA were compared in their ability to sense changes in local Ca2+ concentration, demonstrating preferential sensitivity to Ca2+ originating from intracellular pools (MHA) or from capacitative Ca2+ entry (EHA). Measurements of eNOS activation in intact cells revealed that the eNOS enzymatic activity of EHA was more sensitive to Ca2+ influx via capacitative Ca2+ entry than intracellular release, whereas MHA eNOS activity was more responsive to intracellular Ca2+release. When eNOS activation by CCE was compared with that generated by an equal rise in Ca2+i due to the Ca2+ ionophore ionomycin, a 10-fold greater increase in NO production was found in the former condition. These results demonstrate that EHA and MHA chimera are properly targeted and retain full functions of eNOS and aequorin, and that capacitative Ca2+influx is the principle stimulus for sustained activation of eNOS on the plasma membrane in intact cells.
Traditional combined arms warfare is premised on the assumption that a symmetrical mix of different kinds of units and weapons offers an army the best prospect of achieving optimal results on the ...battlefield. The Israel Defense Forces (IDF) has been accused by critics over the years of essentially abandoning the practice of traditional combined arms warfare before the 1973 Yom Kippur War in favor of a nontraditional (asymmetrical) variant that relied much too heavily on tanks and aircraft. While this criticism certainly has substantial merit, the IDF's reverses in the early days of the war were not due solely to a lack of emphasis on traditional combined arms warfare, but rather also to a "perfect storm" of circumstances that obtained at the outset of hostilities. To its credit, the IDF learned rapidly from its prewar mistakes in force structure and war-fighting doctrine, reverting within days to a traditional approach to the practice of combined arms warfare, at least on the ground. Though not a battlefield panacea by any means, traditional combined arms warfare clearly contributed to the IDF's eventual victory in the war.
Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity ...are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3-4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6-9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 +/- 46% of control levels in hypoxic lungs (P < 0.05) but were decreased by 50 +/- 5 and 60 +/- 11%, respectively, in monocrotaline-treated and fawn-hooded lungs (P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 +/- 1.4 microM; P < 0.05) but not in either monocrotaline-treated (0.7 +/- 0.3 microM) or fawn-hooded (0.45 +/- 0.1 microM) lungs vs. normotensive lungs (0.12 +/- 0.07 microM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-L-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.
Excess aldosterone production contributes to hypertension in both classical hyperaldosteronism and obesity-associated hypertension. Therapies that reduce aldosterone synthesis may lower blood ...pressure.
To compare the safety and efficacy of lorundrostat, an aldosterone synthase inhibitor, with placebo, and characterize dose-dependent safety and efficacy to inform dose selection in future trials.
Randomized, placebo-controlled, dose-ranging trial among adults with uncontrolled hypertension taking 2 or more antihypertensive medications. An initial cohort of 163 participants with suppressed plasma renin (plasma renin activity PRA ≤1.0 ng/mL/h) and elevated plasma aldosterone (≥1.0 ng/dL) were enrolled, with subsequent enrollment of 37 participants with PRA greater than 1.0 ng/mL/h.
Participants were randomized to placebo or 1 of 5 dosages of lorundrostat in the initial cohort (12.5 mg, 50 mg, or 100 mg once daily or 12.5 mg or 25 mg twice daily). In the second cohort, participants were randomized in a 1:6 ratio to placebo or lorundrostat, 100 mg once daily.
The primary end point was change in automated office systolic blood pressure from baseline to study week 8.
Between July 2021 and June 2022, 200 participants were randomized, with final follow-up in September 2022. Following 8 weeks of treatment in participants with suppressed PRA, changes in office systolic blood pressure of -14.1, -13.2, -6.9, and -4.1 mm Hg were observed with 100 mg, 50 mg, and 12.5 mg once daily of lorundrostat and placebo, respectively. Observed reductions in systolic blood pressure in individuals receiving twice-daily doses of 25 mg and 12.5 mg of lorundrostat were -10.1 and -13.8 mm Hg, respectively. The least-squares mean difference between placebo and treatment in systolic blood pressure was -9.6 mm Hg (90% CI, -15.8 to -3.4 mm Hg; P = .01) for the 50-mg once-daily dose and -7.8 mm Hg (90% CI, -14.1 to -1.5 mm Hg; P = .04) for 100 mg daily. Among participants without suppressed PRA, 100 mg once daily of lorundrostat decreased systolic blood pressure by 11.4 mm Hg (SD, 2.5 mm Hg), which was similar to blood pressure reduction among participants with suppressed PRA receiving the same dose. Six participants had increases in serum potassium above 6.0 mmol/L that corrected with dose reduction or drug discontinuation. No instances of cortisol insufficiency occurred.
Among individuals with uncontrolled hypertension, use of lorundrostat was effective at lowering blood pressure compared with placebo, which will require further confirmatory studies.
ClinicalTrials.gov Identifier: NCT05001945.
Two genes,
TSC1 and
TSC2, have been shown to be responsible for tuberous sclerosis (TSC). The detection of loss of heterozygosity of
TSC1 or
TSC2 in hamartomas, the growths characteristically ...occurring in TSC patients, suggested a tumor suppressor function for their gene products hamartin and tuberin. Studies analyzing ectopically modulated expression of
TSC2 in human and rodent cells together with the finding that a homolog of
TSC2 regulates the
Drosophila cell cycle suggest that TSC is a disease of proliferation/cell cycle control. We discuss this question including very recent data obtained from analyzing mice expressing a modulated
TSC2 transgene, and from studying the effects of deregulated
TSC1 expression. Elucidation of the cellular functions of these proteins will form the basis of a better understanding of how mutations in these genes cause the disease and for the development of new therapeutic strategies.
CEP290-associated Leber congenital amaurosis type 10 (LCA10) is a retinal disease resulting in childhood blindness. Sepofarsen is an RNA antisense oligonucleotide targeting the c.2991+1655A>G variant ...in the CEP290 gene to treat LCA10. In this open-label, phase 1b/2 ( NCT03140969 ), 12-month, multicenter, multiple-dose, dose-escalation trial, six adult patients and five pediatric patients received ≤4 doses of intravitreal sepofarsen into the worse-seeing eye. The primary objective was to evaluate sepofarsen safety and tolerability via the frequency and severity of ocular adverse events (AEs); secondary objectives were to evaluate pharmacokinetics and efficacy via changes in functional outcomes. Six patients received sepofarsen 160 µg/80 µg, and five patients received sepofarsen 320 µg/160 µg. Ten of 11 (90.9%) patients developed ocular AEs in the treated eye (5/6 with 160 µg/80 µg; 5/5 with 320 µg/160 µg) versus one of 11 (9.1%) in the untreated eye; most were mild in severity and dose dependent. Eight patients developed cataracts, of which six (75.0%) were categorized as serious (2/3 with 160 µg/80 µg; 4/5 with 320 µg/160 µg), as lens replacement was required. As the 160-µg/80-µg group showed a better benefit-risk profile, higher doses were discontinued or not initiated. Statistically significant improvements in visual acuity and retinal sensitivity were reported (post hoc analysis). The manageable safety profile and improvements reported in this trial support the continuation of sepofarsen development.
During the transformation from a normal to a malignant cell, several mutations are required to bypass the pathways responsible for controlling proliferation. Premalignant cells have acquired some, ...but not all of these mutations and consequently have not yet attained a malignant phenotype characterized by tumor formation
in vivo
. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in malignant cells while sparing normal ones and is currently being considered as adjuvant therapy for various human malignancies. Whether TRAIL is effective in inducing apoptosis in premalignant cells is unclear, however. We studied the effect of TRAIL on two human premalignant cell lines the SV7tert and HA1E cells. Both cell lines had been immortalized by the addition of simian virus 40 large T antigen and the telomerase subunit hTERT, but had not been transformed into malignant cells. TRAIL initiated apoptosis by activating both the mitochondrial-independent and -dependent apoptotic pathways in both cell lines at relatively low doses whereas it had no effect on normal human pulmonary artery smooth muscle cells even at high doses. These results suggest that TRAIL can induce apoptosis in premalignant cells and suggests a novel therapy for the treatment of premalignant lesions
in vivo
.
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