Lucie H. Clapp

Terlipressin for norepinephrine-resistant septic shock.

Norepinephrine-resistant hypotension when associated with septic shock has a high rate of mortality, which might possibly be reduced by infusion of low-dose vasopressin. However, rebound hypotension often arises after treatment is stopped, and the drug usually has to be administered for several days. We report use of terlipressin, a long-acting vasopressin analogue, in eight patients with septic shock who did not respond to corticosteroids and methylene blue. A significant rise in blood pressure that lasted for at least 5 h was seen in all patients after a single bolus, allowing reduction or cessation of norepinephrine administration in seven patients. We were able to discharge four patients from intensive care subsequently. Terlipressin seems to be an effective rescue therapy, which is able to restore blood pressure in patients with catecholamine-resistant septic shock, without obvious complication.

The large-conductance Ca2+-activated K+ channel is essential for innate immunity

Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BKCa) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BKCa channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the α-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BKCa channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.

Vasopressin: mechanisms of action on the vasculature in health and in septic shock.

Background: Vasopressin is essential for cardiovascular homeostasis, acting via the kidney to regulate water resorption, on the vasculature to regulate smooth muscle tone, and as a central neurotransmitter, modulating brainstem autonomic function. Although it is released in response to stress or shock states, a relative deficiency of vasopressin has been found in prolonged vasodilatory shock, such as is seen in severe sepsis. In this circumstance, exogenous vasopressin has marked vasopressor effects, even at doses that would not affect blood pressure in healthy individuals. These two findings provide the rationale for the use of vasopressin in the treatment of septic shock. However, despite considerable research attention, the mechanisms for vasopressin deficiency and hypersensitivity in vasodilatory shock remain unclear. Objective: To summarize vasopressin’s synthesis, physiologic roles, and regulation and then review the literature describing its vascular receptors and downstream signaling pathways. A discussion of potential mechanisms underlying vasopressin hypersensitivity in septic shock follows, with reference to relevant clinical, in vivo, and in vitro experimental evidence. Data Source: Search of the PubMed database (keywords: vasopressin and receptors and/or sepsis or septic shock) for articles published in English before May 2006 and manual review of article bibliographies. Data Synthesis and Conclusions: The pathophysiologic mechanism underlying vasopressin hypersensitivity in septic shock is probably multifactorial. It is doubtful that this phenomenon is merely the consequence of replacing a deficiency. Changes in vascular receptors or their signaling and/or interactions between vasopressin, nitric oxide, and adenosine triphosphate‐dependent potassium channels are likely to be relevant. Further translational research is required to improve our understanding and direct appropriate educated clinical use of vasopressin.

Smooth Muscle Proliferation and Role of the Prostacyclin (IP) Receptor in Idiopathic Pulmonary Arterial Hypertension

RATIONALE Prostacyclin analogs, used to treat idiopathic pulmonary arterial hypertension (IPAH), are assumed to work through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, although the potential to signal through peroxisome proliferator-activated receptor-γ (PPARγ) exists. OBJECTIVES IP receptor and PPARγ expression may be depressed in IPAH. We wished to determine if pathways remain functional and if analogs continue to inhibit smooth muscle proliferation. METHODS We used Western blotting to determine IP receptor expression in peripheral pulmonary arterial smooth muscle cells (PASMCs) from normal and IPAH lungs and immunohistochemistry to evaluate IP receptor and PPARγ expression in distal arteries. MEASUREMENTS AND MAIN RESULTS Cell proliferation and cAMP assays assessed analog responses in human and mouse PASMCs and HEK-293 cells. Proliferative rates of IPAH cells were greater than normal human PASMCs. IP receptor protein levels were lower in PASMCs from patients with IPAH, but treprostinil reduced replication and treprostinil-induced cAMP elevation appeared normal. Responses to prostacyclin analogs were largely dependent on the IP receptor and cAMP in normal PASMCs, although in IP(-/-) receptor cells analogs inhibited growth in a cAMP-independent, PPARγ-dependent manner. In IPAH cells, antiproliferative responses to analogs were insensitive to IP receptor or adenylyl cyclase antagonists but were potentiated by a PPARγ agonist and inhibited (∼ 60%) by the PPARγ antagonist GW9662. This coincided with increased PPARγ expression in the medial layer of acinar arteries. CONCLUSIONS The antiproliferative effects of prostacyclin analogs are preserved in IPAH despite IP receptor down-regulation and abnormal coupling. PPARγ may represent a previously unrecognized pathway by which these agents inhibit smooth muscle proliferation.

Effects of the adenylyl cyclase inhibitor SQ22536 on iloprost‐induced vasorelaxation and cyclic AMP elevation in isolated guinea‐pig aorta

The stable prostacyclin analogue, iloprost relaxes a variety of blood vessels and increases cyclic AMP, although the relationship between adenosine 3′ : 5′‐cyclic monophosphate (cyclic AMP) and vasorelaxation remains unclear. We therefore investigated the effect of the adenylyl cyclase inhibitor, 9‐(tetrahydro‐2‐furanyl)‐9H‐purin‐6‐amine (SQ22536) on iloprost‐mediated relaxation and cyclic AMP elevation in endothelium‐denuded aortic strips. Iloprost (1–1000 nM) caused a concentration‐dependent inhibition of phenylephrine (1–6 μM) contractions, the responses being unaffected by pre‐incubation with SQ22536 (100 μM) for 30 min. In other experiments 60 nM iloprost caused a 64% inhibition of phenylephrine contractions concomitant with a 3 fold rise in cyclic AMP. SQ22536 completely abolished the iloprost‐induced elevation in cyclic AMP while having no significant effect on relaxation. Our results therefore strongly suggest that cyclic AMP‐independent pathways are responsible for the vasorelaxant effects of iloprost in guinea‐pig aorta.

Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist.

The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated.

Calcium modulation of vascular smooth muscle ATP-sensitive K+ channels - Role of protein phosphatase-2B

ATP-sensitive K+ (KATP) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP]i) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that KATP channels are insensitive to Ca2+, although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca2+ on whole-cell KATP current in rat aortic smooth muscle cells recorded in a physiological [ATP]i and K+ gradient. Under control recording conditions, cells had a resting potential of ≈−40 mV when bathed in 1.8 mmol/L Ca2+. The KATP channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP]i from 3 to 0.1 mmol/L hyperpolarized cells to ≈−60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca2+ levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca2+]i showed that KATP current was maximally activated at 10 nmol/L [Ca2+]i and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca2+-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by ≈20 to 25 mV. We report for the first time that KATP channels can be modulated by Ca2+ at physiological [ATP]i and conclude that modulation occurs via protein phosphatase type 2B.

Evidence that Ca2+-activated K+ channels play a major role in mediating the vascular effects of iloprost and cicaprost

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.

Temporal variation in endotoxin-induced vascular hyporeactivity in a rat mesenteric artery organ culture model

Endotoxin‐induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro. Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46 h in the presence or absence of bacterial lipopolysaccharide (LPS; 1 – 100 μg ml−1). Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L‐NAME (300 μM), aminoguanidine (AMG; 400 μM) 1400W (10 μM) and GW273629 (10 μM); the guanylyl cyclase inhibitor, ODQ (3 μM); the COX‐2 inhibitor, NS‐398 (10 μM). Contractile responses to the thromboxane A2 mimetic, U46619 were also assessed. In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h. L‐NAME and AMG fully reversed hyporeactivity at 6 h, whereas they were only partially effective at 20 h and not at all at 46 h. In contrast partial reversal of peak contraction was observed with 1400W (62% at 46 h), GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX‐2 inhibition produced no reversal. In contrast to PE, contractions to U46619 were substantially less affected by LPS. We describe a well‐characterized reproducible model of LPS‐induced hyporeactivity, which is largely mediated by the NO‐cyclic GMP‐dependent pathway. Importantly, long‐term (2‐day) production of NO via iNOS is demonstrated. Moreover, conventional doses of L‐NAME and AMG became increasingly ineffective over time. Thus, the choice of inhibitor merits careful consideration in long‐term models.

The pore‐forming subunit of the KATP channel is an important molecular target for LPS‐induced vascular hyporeactivity in vitro

1 ATP‐sensitive K+ (KATP) channel activation is implicated in the vascular hyporeactivity occurring in septic shock. However, channel inhibition with the sulphonylurea receptor (SUR) antagonist, glibenclamide (Glib) fails to reverse lipopolysaccharide (LPS)‐induced vascular hyporeactivity in vitro. We investigated whether inhibitors that act by binding to the KATP channel pore could be effective. 2 Ring segments of endothelium‐intact rat mesenteric artery were incubated with LPS in culture media for either 6 or 20 h before contractile responses to phenylephrine were assessed in the absence or presence of KATP channel inhibitors. 3 The pore‐forming subunit inhibitors barium chloride (BaCl2; 300 μM) and PNU‐37883A (1 μM) significantly reversed hyporeactivity at both time points, although less so at 20 h. In contrast, the SUR inhibitors, Glib (10 μM), tolbutamide (Tolb) (1 mM) and PNU‐99963 (1 μM) were ineffective. In LPS‐incubated tissues, Glib and Tolb antagonised contractions to the thromboxane A2 mimetic, U46619 (9,11‐dideoxy‐9α, 11α‐methanoepoxy prostaglandin F2α) (10−7 M), whereas the pinacidil‐derived inhibitor, PNU‐99963, did not. 4 Contractions to 60 mM KCl were unaffected by LPS at 6 h, but were significantly depressed by LPS at 20 h, suggesting that K+‐channel‐independent pathways contribute to hyporeactivity at the later time point. 5 The inducible nitric oxide synthase (iNOS) inhibitor, 1400 W (10 μM) and Tolb inhibited the production of nitrite induced by LPS, whereas BaCl2 and PNU‐37883A had no effect. 6 In conclusion, KATP channels contribute to LPS‐induced vascular hyporeactivity via the iNOS pathway in rat mesenteric artery. The effectiveness of pore inhibitors over SUR inhibitors of the KATP channel suggests altered SUR function following LPS administration, which cannot be explained by thromboxane receptor inhibition.