Comparison of PEAK PlasmaBlade™ to conventional diathermy in abdominal-based free-flap breast reconstruction surgery-A single-centre double-blinded randomised controlled trial.

BACKGROUND: Electrosurgery makes dissection with simultaneous haemostasis possible. The produced heat can cause injury to the surrounding tissue. The PEAK PlasmaBlade™(PPB) is a new electrosurgery device which may overcome this by having the ability to operate on a lower temperature, therefore reducing collateral thermal damage. METHOD: A single-centre, double-blinded, randomised controlled trial (RCT) was conducted which included 108 abdominal-based free-flap breast reconstruction patients who had their flap raise performed using either the PPB (n = 56) or the conventional diathermy (n = 52). Data were collected during their in-patient stay and out-patient appointments. The primary outcome value was the number of days the abdominal drains were required. RESULTS: Baseline characteristics were similar between the groups, except a significantly lower flap weight in the PPB group. The median number of days the drains were required did not differ significantly (p = 0.48; 6.0 days for the diathermy and 5.0 days for the PPB). The total drain output (p = 0.68), the inflammatory cytokine in the drain fluid (p>0.054) and complications (p>0.24) did not differ significantly between the two groups. At the 2-week follow-up appointment, there was a trend towards less abdominal seromas on abdominal ultrasound (p = 0.09) in the PPB group which were significantly smaller (p = 0.04). CONCLUSION: The use of the PPB did not result in a significant reduction of drain requirement, total drain output or inflammatory cytokines but did reduce the size of seroma collections at the 2-week follow-up appointment. Therefore, the use of the PPB device could reduce early seroma formation after drain removal.

What role do pharmaceuticals play in the treatment of Peyronie's disease and is there a need for new emerging drugs?

INTRODUCTION: Finding novel medical treatment for Peyronie's disease (PD) has suffered from similar limitations and difficulties as other fibrotic diseases.Areas covered: Underlying fibrosis, there is a vastly complex intertwining of several pathways. Focusing on a single target during antifibrotic drug development has not led to the development of many efficacious drugs, especially in PD. Inhibiting one cog in this large machinery usually leads to activation of compensatory mechanisms.Expert opinion: Novel strategies in drug discovery such as phenotypical drug screening and gene expression profiling technologies could provide a solution for this impasse.

Pathophysiology of diabetic erectile dysfunction: potential contribution of vasa nervorum and advanced glycation endproducts.

Erectile dysfunction (ED) due to diabetes mellitus remains difficult to treat medically despite advances in pharmacotherapeutic approaches in the field. This unmet need has resulted in a recent re-focus on the pathophysiology, in order to understand the cellular and molecular mechanisms leading to ED in diabetes. Diabetes-induced ED is often resistant to PDE5 inhibitor treatment, thus there is a need to discover targets that may lead to novel approaches for a successful treatment. The aim of this brief review is to update the reader in some of the latest development on that front, with a particular focus on the role of impaired neuronal blood flow and the formation of advanced glycation endproducts.

GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis.

5-hydroxyindalpine, an agonist at the putative 5-HT receptor, has no activity on human recombinant monoamine receptors but accelerates distension-induced peristalsis in mouse isolated colon.

Although the putative 5-HT(1P) receptor has been implicated to have a role in peristalsis, experiments which suggest this function are preliminary or have measured only components of the reflex. We have, therefore, further characterized a reported agonist at this receptor (5-hydroxyindalpine; 5-OHIP) and investigated the effects of 5-OHIP and 5-hydroxytrytophan-dipeptide (5-HTP-DP), a reported 5-HT(1P) receptor antagonist, on distension-induced peristalsis in mouse colon. The effects of 5-OHIP on intracellular calcium, cyclic adenosine monophosphate concentrations or GTPgammaS binding were measured in cell lines expressing human recombinant 5-HT(1A, 1B, 1D, 2A, 2B, 2C, 3, 4, 6, 7) and alpha(1A), alpha(1B), D(1), D(2), D(3), H(1), H(3) receptors. The effects of 5-OHIP and 5-HTP-DP on peristalsis were assessed by measuring changes in frequency and times to reach threshold of peristaltic contractions, as well as the threshold and maximum pressures of each peristaltic stroke. 5-hydroxyindalpine (1 nmol L(-1)-10 micromol L(-1)) had no significant activity at any of the receptors studied. However, 5-OHIP (0.1 nmol L(-1)-1 micromol L(-1)) concentration-dependently increased the frequency of peristalsis (EC(50) = 4.4 nmol L(-1)) and reduced the time taken to reach threshold and threshold pressure, without altering maximum pressures. The maximum effect of 5-OHIP was at 1 micromol L(-1) (68.0 +/- 14.5% increase in frequency); 10 micromol L(-1) decreased peristalsis. 5-hydroxytrytophan-dipeptide (1-300 nmol L(-1)) also increased the frequency of peristalsis and prevented the actions of 5-OHIP. The higher concentration (1 micromol L(-1)) transiently inhibited peristalsis and after recovery, prevented the actions of 5-OHIP but not the excitatory activity of the cholinesterase inhibitor neostigmine. In summary, the present data demonstrate an interaction of '5-HT(1P)-ligands' with the peristaltic reflex. However, the absence of an effect of 5-OHIP on a range of different monoamine receptors continues to highlight the need to investigate the identity of the putative 5-HT(1P) receptor.

Synergy between 5-HT4 receptor activation and acetylcholinesterase inhibition in human colon and rat forestomach.

5-Hydroxytryptamine (5-HT4) receptor agonists increase gastrointestinal (GI) motility by enhancing enteric acetylcholine release which is then metabolized by acetylcholinesterase (AChE) to inactive metabolites. As both AChE inhibitors and, more usually, 5-HT4 receptor agonists are used to increase GI motility, an understanding of how these two different types of drugs might interact becomes of great importance. Our aim was to investigate the hypothesis that the effect of AChE inhibition will synergise with the ability of 5-HT4 receptor agonism to increase cholinergic activity, leading to an effect greater than that evoked by each action alone. We tested the activity of the 5-HT4 receptor agonist, prucalopride (10 nmol L(-1)-30 micromol L(-1)) and an AChE inhibitor, neostigmine (1 nmol L(-1)-10 micromol L(-1)) on cholinergically mediated contractions elicited by electrical field stimulation of human isolated colon circular muscle and rat isolated forestomach longitudinal strips. The experiments with human colon were performed in the presence of an inhibitor of nitric oxide synthase (N(omega)-nitro-l-arginine methyl ester, 300 micromol L(-1)). Prucalopride and neostigmine both enhanced cholinergic contractions in both tissues. The effect of prucalopride was inhibited in both tissues by SB-204070, a 5-HT4 receptor antagonist. In the presence of a minimum effective concentration of neostigmine (30 nmol L(-1)) and a submaximum concentration of prucalopride (3 micromol L(-1)) the enhancement of contractions was greater than either compound alone in both tissues. These data demonstrate that the combination of prucalopride and neostigmine potentiate cholinergic contractions more than their arithmetic sum of their individual values. The results suggest that a synergy between 5-HT4 receptor agonism and AChE inhibition could be established pharmacologically which could be utilized as a novel prokinetic approach to functional GI disorders.

5-HT4 receptor agonists enhance both cholinergic and nitrergic activities in human isolated colon circular muscle.

Previous studies have demonstrated mixed inhibitory and facilitatory effects of 5-hydroxytryptamine-4 (5-HT(4)) receptor agonists on electrical field stimulation (EFS)-induced responses in human isolated colon. Here we report three types of responses to EFS in human isolated colon circular muscle: monophasic cholinergic contraction during EFS, biphasic response (nitrergic relaxation during EFS followed by cholinergic contraction after termination of EFS) and triphasic response (cholinergic contraction followed by nitrergic relaxation during EFS and a tachykininergic contraction after EFS). The effects of two 5-HT(4) receptor agonists, prucalopride and tegaserod were then investigated on monophasic responses only. Each compound inhibited contractions during EFS in a concentration-dependent manner. In the presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) however, prucalopride and tegaserod enhanced the contractions in a concentration-dependent manner. In strips where the tone was elevated with substance-P and treated with scopolamine, EFS-induced relaxations were enhanced by the two agonists. The above observed effects by the two agonists were abolished by 5-HT(4) receptor antagonist SB-204070. The two agonists did not alter the tone raised by substance-P in the presence of scopolamine and l-NAME and did not affect carbachol-induced contractions in the presence of tetrodotoxin. These results suggest that in the circular muscle of human colon, 5-HT(4) receptor agonists simultaneously facilitate the activity of neurones which release the inhibitory and excitatory neurotransmitters, nitric oxide and acetylcholine respectively.

Investigation of cavernosal smooth muscle dysfunction in low flow priapism using an in vitro model.

The effects of hypoxia (pO2: 50 mmHg), acidosis (pH: 6.9) or glucopenia (absence of glucose) in vitro on the tone of the rabbit corpus cavernosum were investigated. The recovery of smooth muscle contractility following exposure to these conditions was also assessed. Hypoxia, acidosis or glucopenia alone or in combination showed a sustained reduction in the tone. Reperfusion of tissue strips showed complete recovery of smooth muscle tone for all conditions except when hypoxia and glucopenia were combined or when hypoxia, glucopenia and acidosis were used in combination. Incomplete recovery of tone was associated with a significant reduction in tissue ATP concentrations and an increase in the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive nuclei. This indicates that following reversal of hypoxia, acidosis and glucopenia, failure of conventional alpha-adrenergic agonists to produce tumescence in low flow priapism is associated with irreversible smooth muscle cell dysfunction, which is linked to ATP reduction and smooth muscle cell death.

Point of NO return for nitrergic nerves in diabetes: a new insight into diabetic complications.

Aberrations in nitrergic neurotransmission, due to a decrease in neuronal nitric oxide (NO) synthase (nNOS) protein, play an important role in the pathogenesis of autonomic neuropathy in diabetes. Until recently the mechanism of the decrease in nNOS protein content in nitrergic nerves during diabetes was debated. Two different views were prevailing, one attributing the nNOS decrease to nitrergic nerve degeneration, the other to an alteration in nNOS expression. Our recent study in which we showed that nitrergic nerves undergo a degenerative process in two phases might bring a solution to this debate. Our model suggests that, in the early stages of diabetes, nNOS expression is decreased in the nitrergic axons while nNOS levels are unaffected in the cell bodies, most probably due to a defect in axonal transport. This decrease is reversible with insulin treatment. As the diabetes progresses, nNOS starts to accumulate in the cell bodies since it cannot be transported down to the axons. Increased nNOS protein and NO production coincide with accumulation of advanced glycation endproducts (AGEs) in the blood and tissues. Synergistic action of AGEs and endogenous NO leads to increased oxidative stress within the cell bodies, resulting in apoptosis. This degenerative phase of nitrergic neuropathy is not reversible with insulin treatment. This suggests a point of no return for autonomic nerves after which the degenerative changes become irreversible. Future therapeutic approaches could target the defective axonal transport and prevention of AGEs accumulation before this point of no return. In the later stages, reduction of AGEs, replenishment of lost nitrergic neurons and restoration of function are putative therapeutic targets.

Functional evidence for nitrergic neurotransmission in a human clitoral corpus cavernosum: a case study.

Although the presence of neuronal nitric oxide synthase (nNOS) has been demonstrated in human clitoral corpus cavernosum, functional evidence for the nitrergic neurotransmission as a nonadrenergic noncholinergic (NANC) transmitter has been limited to animal studies. Here we show that electrical field stimulation elicited reproducible NANC relaxation responses in a clitoral corpus cavernosum, obtained from a 38-y-old woman undergoing clitoral reduction surgery. These relaxation responses were abolished by an inhibitor of NO synthase or tetrodotoxin proving that they were nitrergic in nature and neuronal in origin. Large and small calibre nitrergic nerves were demonstrated with immunohistochemistry using nNOS antibody, which were also immunostained with cholinergic nerve markers. nNOS expression was confirmed using Western blotting. This is the first report to show that NO produced by nNOS within the cholinergic-nitrergic nerves is responsible for the NANC relaxation responses in a human clitoral corpus cavernosum.

NCX-911, a novel nitric oxide-releasing PDE5 inhibitor relaxes rabbit corpus cavernosum in the absence of endogenous nitric oxide.

Phosphodiesterase type 5 (PDE5) inhibitors have reduced efficacy in treating erectile dysfunction (ED) in conditions where there is a lack of endogenous nitric oxide (NO). Therefore, NO-releasing PDE5 inhibitors have been developed. Here we report the effect of such a compound, NCX-911, on the tone and nitrergic relaxations of rabbit corpus cavernosum in the absence or presence of a NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 500 microM). NCX-911 was found to be as potent as sildenafil at inducing relaxation of rabbit cavernosum (EC(50) values 997.8+/-195.7 and 1000.5+/-140.8 nM, respectively). The potency of NCX-911 was not altered, but that of sildenafil decreased five-fold in the presence of L-NAME (EC(50) values 1281.2+/-268.3 and 4959.1+/-882.1, nM respectively, P<0.001 for sildenafil). Both compounds potentiated nitrergic relaxations with similar potencies. These results suggest that NO-releasing PDE5 inhibitors could potentially be more useful than PDE5 inhibitors in the treatment of ED in conditions where there is a lack of endogenous NO.

Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes.

AIMS/HYPOTHESIS: We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression. METHODS: Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose. RESULTS: AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase. CONCLUSIONS/INTERPRETATION: The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.

Current oral treatments for erectile dysfunction.

Erectile dysfunction (ED) is defined as the inability to achieve and maintain a penile erection adequate for satisfactory sexual intercourse. It is a significant male health problem of global dimensions affecting approximately 150 million men worldwide. A broad range of options are currently available for the management of ED. They include oral agents (phosphodiesterase 5 inhibitors, dopamine agonists and alpha-receptor blocking drugs), intracavernosal injection (papaverine, phentolamine, prostaglandin E1, vasoactive intestinal peptide), transurethral vasoactive agents (prostaglandin E1), vacuum erection devices, vascular surgery and penile prostheses. Here we review the physiology of penile erection and the currently available oral preparations. In addition, novel therapeutic strategies to improve erectile function are discussed.

Human and rabbit cavernosal smooth muscle cells express Rho-kinase.

Rho-kinase is an enzyme involved in the Ca2+-sensitizing pathway in smooth muscle cells. Inhibition of this enzyme has been recently demonstrated to elicit penile erection by relaxing cavernosal smooth muscle. We aimed to investigate the presence and activity of Rho-kinase in human cavernosal smooth muscle. Primary culture of smooth muscle cells from human and rabbit penile corpus cavernosum was developed, and cells showed characteristic myocyte morphology and alpha-actin immunoreactivity. The presence of Rho-kinase was demonstrated by indirect immunofluorescence and Western blotting. A specific inhibitor of Rho-kinase, Y-27632, inhibited in a concentration-dependent manner the kinase activity of the protein immunoprecipitated with anti-Rho-kinase antibody. These results demonstrate for the first time expression and activity of Rho-kinase in human penile cavernosal smooth muscle cells and suggest that these cells can provide a cellular model for the study of enzymes involved in Ca2+-sensitizing pathways.

Y-27632, an inhibitor of Rho-kinase, antagonizes noradrenergic contractions in the rabbit and human penile corpus cavernosum.

We have examined the effect of an inhibitor of Rho-kinase, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), on the contractions elicited by noradrenergic nerve stimulation and by phenylephrine in the human and rabbit penile corpus cavernosum. In both tissues, after treatment with scopolamine (10 microM) and N(G)-nitro-L-arginine methyl ester (L-NAME; 300 microM), electrical field stimulation (EFS) elicited noradrenergic contractions. These contractions were inhibited by Y-27632 in a concentration-dependent manner. The compound caused concentration-dependent relaxation of phenylephrine-contracted tissues, which were treated with scopolamine (10 microM), guanethidine (10 microM) and L-NAME (300 microM). These results suggest that Rho-kinase is involved in noradrenergic contractile pathway in the cavernosal smooth muscle of the penis.

Nitrergic-noradrenergic interaction in penile erection: a new insight into erectile dysfunction.

Penile erection is regulated by two opposing systems: noradrenergic (anti-erectile) and nitrergic (pro-erectile) neurotransmission. Noradrenaline released from sympathetic nerves causes contraction of the blood vessels and smooth muscle of the penile corpus cavernosum, thus leading to detumescence of the penis. Nitric oxide (NO) released from nitrergic nerves causes relaxation of the smooth muscle of the corpus cavernosum, thus allowing engorgement of blood into the cavernous space and leading to erection. Nitrergic neurotransmission is known to modulate noradrenergic responses. We have recently shown that the degree of this modulation varies among species. In the human corpus cavernosum, noradrenergic responses are under nitrergic control, such that even pharmacological concentrations of noradrenaline fail to show an effect when nitrergic neurotransmission is operating. This situation is similar in the monkey and rabbit, where nitrergic neurotransmission does not merely modulate but actually controls the sympathetic responses; however it differs in the rat, mouse and dog where the sympathetic system is predominant. Our recent work has demonstrated that the interaction between the two systems occurs in the smooth muscle, suggesting a physiological antagonism. Our observations suggest that the key element in this interaction is intracellular calcium in the smooth muscle. The nitrergic pathway causes a decrease in intracellular calcium concentrations thus leading to relaxation of the smooth muscle. Noradrenergic stimulation, in contrast, elicits an increase in the intracellular calcium concentrations thus leading to a contraction. The neuronal pathway which controls the concentrations of intracellular calcium in the smooth muscle determines the dominance of that pathway over the other. Nitrergic dominance over noradrenergic system in the human corpus cavernosum also suggests a key role for this interaction in the pathophysiology of erectile dysfunction. Indeed, a nitrergic-noradrenergic imbalance in favor of the noradrenergic system has been implicated in penile tissues from patients with erectile dysfunction. However, the mechanism of this imbalance is not fully understood. In addition, since the present study has demonstrated that phosphodiesterase type V inhibitors can enhance and prolong the nitrergic control of noradrenergic responses, such compounds may have therapeutic potential in impotence, where defective nitrergic transmission is accompanied by increased noradrenergic activity.

Selective nitrergic neurodegeneration in diabetes mellitus - a nitric oxide-dependent phenomenon.

1. In vitro and in vivo studies have demonstrated a dysfunctional nitrergic system in diabetes mellitus, thus explaining the origin of diabetic impotence. However, the mechanism of this nitrergic defect is not understood. 2. In the penises of streptozotocin (STZ)-induced diabetic rats, here, we show by immunohistochemistry that nitrergic nerves undergo selective degeneration since the noradrenergic nerves which have an anti-erectile function in the penis remained intact. 3. Nitrergic relaxation responses in vitro and erectile responses to cavernous nerve stimulation in vivo were attenuated in these animals, whereas noradrenergic responses were enhanced. 4. Activity and protein amount of neuronal nitric oxide synthase (nNOS) were also reduced in the penile tissue of diabetic rats. 5. We, thus, hypothesized that NO in the nitrergic nerves may be involved in the nitrergic nerve damage, since only the nerves which contain neuronal NO synthase underwent degeneration. 6. We administered an inhibitor of NO synthase, N(G)-nitro-L-arginine methyl ester (L-NAME), in the drinking water of rats for up to 12 weeks following the establishment of diabetes with STZ. 7. Here we demonstrate that this compound protected the nitrergic nerves from morphological and functional impairment. Our results show that selective nitrergic degeneration in diabetes is NO-dependent and suggest that inhibition of NO synthase is neuroprotective in this condition.

Visualization of nitric oxide formation in cell cultures and living tissue.

We have visualized nitric oxide (NO) released from cell cultures and living tissue. NO was visualized by a reaction with luminol and hydrogen peroxide to yield photons which were counted using a microscope coupled to a photon counting camera. Murine macrophages were activated with interferon-gamma (IFN-gamma) and endotoxin (LPS). Cultured endothelial cells were stimulated with bradykinin, and neurones in the guinea-pig myenteric plexus and the rabbit hypogastric nerve trunk were electrically stimulated. There was a marked increase in photons emitted from the cultured cells as well as from the living tissues during stimulation. The stimulation-induced photon emission was markedly reduced by inhibition of nitric oxide synthase (NOS); removal of L-arginine from the medium also decreased photon counts. The present method allowed integration times in the order of minutes to improve signal-to-noise ratio. However, the high sensitivity of this method also makes it possible to generate an image in seconds, allowing the production of real time films. Photon emission was enhanced under conditions known to increase NO production, and diminished in the presence of NO inhibitors. Thus, this method has demonstrated specificity for the L-arginine:NO pathway from a wide range of biological sources such as cultured cells and living tissues, and has the potential for real time imaging of NO formation, with high temporal and spatial resolution.

Fatty acylation of phospholipase D1 on cysteine residues 240 and 241 determines localization on intracellular membranes.

We have reported previously that phospholipase D1 (PLD1) is labeled specifically with [(3)H]palmitate following transient expression and immunoprecipitation and that this modification appeared important both for membrane localization and catalytic activity. In this work we identify by mutagenesis that the acylation sites on PLD1 are cysteine residues 240 and 241, with the cysteine at position 241 accounting for most but not all of the modification. Replacement of both cysteine residues with either serines or alanines resulted in a mutant protein that contained undetectable [(3)H]palmitate. In comparison with the wild type protein, the double mutant showed reduced catalytic activity in vivo, whereas its activity in vitro was unchanged. In addition, the localization of the double mutant was altered in comparison with the wild type protein, whereas wild type PLD1 is primarily on intracellular membranes and on punctate structures, the double mutant was on plasma membrane. Because cysteines 240 and 241 lie within a putative pleckstrin homology domain of PLD1, it is likely that fatty acylation on these residues modulates the function of the PLD1 pleckstrin homology domain.