Involvement of TLR6 in the induction of COX-2, PGE2 and IL-10 in macrophages by lipids from virulent S2P and attenuated R1A Babesia bovis strains.

Toll like receptors (TLRs) are involved in the modulation of diverse host genes expression through a complex network of signalling events that allow for an appropriate response to a microbial pathogen. In the present work we used TLR6KO mice in order to study the role of TLR6 in the immune discrimination of lipids from two Babesia bovis strains, attenuated R1A (LA) and virulent S2P (LV), and the consequent macrophage activation. We demonstrated that TLR6 is required for lipid body induction in murine peritoneal macrophages by both LA and LV. Interestingly, as regards IL-10 and COX-2/PGE2 pathway induction by LA and LV, we observed differences in the biological effects produced by these lipid extracts. Our results indicate a role of TLR6 in the down-modulation of these immunoregulators only in the case of LA, whereas this receptor was not implicated in pro-inflammatory TNFα, IL-6 and KC release induced by LA. Remarkably, LV did not exert the down-modulatory effect observed for LA, supporting the notion that LA and LV possess different lipid composition that could correlate with the polar pathogenic effect of both B. bovis strains.

Babesia bovis: lipids from virulent S2P and attenuated R1A strains trigger differential signalling and inflammatory responses in bovine macrophages.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Lipids from attenuated and virulent Babesia bovis strains induce differential TLR2-mediated macrophage activation.

Babesia bovis is an intraerythrocytic apicomplexan protozoa of cattle that causes an acute infection with parasite persistence. Babesiosis limitation depends on macrophages, essential effector cells of the host innate defense, which generate inflammatory cytokines and nitric oxide. Herein, we report quantitative differences in the lipid composition of merozoites from two B. bovis strains with polar behaviour: attenuated R1A and virulent S2P. Accordingly, we observed a distinct inflammatory response induced by the total lipids of R1A (L(A)) and S2P (L(V)) in murine peritoneal macrophages. L(A) and particularly its fractions phosphatidic acid and phosphatidylserine+phosphatidylinositol (PS+PI), produced a strong activation of these cells with lipid body formation, cyclooxygenase-2 expression and pro-inflammatory TNFalpha, IL-6 and KC secretion. Although L(V) did not activate these cells, the corresponding PS+PI fraction induced TNFalpha, IL-6 and KC release. Therefore, these facts might be suggesting the presence of an inhibitor in L(V). Furthermore, the employment of wild type and toll like receptor 2 knockout (TLR2KO) mice allowed us to demonstrate that macrophage activation by the stimulating lipid fractions was mediated through TLR2. Interestingly, only L(A) activated the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Inhibitory studies employing UO126, indicated that the ERK pathway was required for TNFalpha, IL-6 and KC release. In conclusion, the absence of inflammatory response observed with the lipids of S2P virulent strain could constitute an evasion mechanism of the innate immune response enabling parasite establishment in the host.

Phospholipase A1 from Trypanosoma cruzi infective stages generates lipid messengers that activate host cell protein kinase c.

Here we have studied phospholipase A1 (Plase A1) from Trypanosoma cruzi infective stages and it's possible role regarding the interaction with mammalian host cells. Plase A1 was mainly detected as a membrane-bound activity in the infective amastigote and trypomastigote stages, being remarkably higher with respect to the non-infective epimastigotes. It is noteworthy that only the infective stages secreted Plase A1. Moreover, along the differentiation process from epimastigotes into metacyclic trypomastigotes, the secreted enzyme activity increased simultaneously with the appearance of metacyclic forms, as expected. Since this enzyme is predominantly membrane-associated and secreted by the infective stages, Vero cell lipid profile modifications were analysed after interaction with either intact infective parasites or purified T. cruzi Plase A1. Significant changes in Vero cell lipid composition were observed, with the appearance of free fatty acids, diacylglycerol and lysophosphatidylcholine. Concomitantly with the generation of second lipid messengers, host cell protein kinase C activation was demonstrated. These results indicate that T. cruzi Plase A1 could play a critical role in the early events of parasite-host cell interaction that precede invasion.

Novel cysteine proteinase in Trypanosoma cruzi metacyclogenesis.

With the aim to study proteinases released to the culture medium during Trypanosoma cruzi metacyclogenesis, the presence of cysteine proteinases (CPs) was analysed in culture supernatants obtained throughout the differentiation induced by stimulation of epimastigotes with Triatoma infestans hindgut homogenate. In SDS-gelatin containing gels, an important endopeptidase activity with apparent molecular weight range between 97 and 116 kDa was encountered at pH 6, which was abolished by the specific cysteine proteinase inhibitor E-64 and TLCK, but not by pepstatin, 1,10 phenantroline or PMSF. This novel CP, named TcCPmet, showed affinity to cystatin-Sepharose, denoting its thiol-proteinase character as well as to ConA-Sepharose, indicating it contains N-linked oligosaccharides. However, it presented a different elution pattern on ConA-Sepharose than cruzipain and, in addition, it was not recognized by anti-cruzipain serum, facts that strongly suggest the different nature of both CPs. Moroever, evidence is presented indicating that TcCPmet was able to hydrolyse the same chromogenic peptides as cruzipain at optimal alkaline pH values, although with a different order of effectiveness. Our results indicate the presence of a novel CP secreted by metacyclic trypomastigotes and reinforces the important role of these enzymes in metacyclogenesis.

Temperature acclimation of Trypanosoma cruzi epimastigote and metacyclic trypomastigote lipids.

This study examines the changes in cellular lipids that take place when Trypanosoma cruzi epimastigotes and metacyclic trypomastigotes are transferred from 28 to 37 degrees C. We found a rise in the sterol to phospholipid ratio, as well as in the triacylglycerol and steryl ester cellular content in T. cruzi epimastigotes. In addition, saturated to unsaturated fatty acid ratios in phospholipids increase. This latter effect appears to be due to two concurrent processes. Firstly, fatty acyl delta9 and, especially, delta12 desaturations are significantly diminished at 37 degrees C. Secondly, triacylglycerols and steryl esters undergo changes in their fatty acyl composition opposite to those simultaneously observed in phospholipids, i.e. the ratio of saturated to unsaturated fatty acids markedly decreases. Similar alterations in each of the lipid classes and in the fatty acid composition of polar and neutral lipids were found in cultured metacyclic trypomastigotes on exposure to the same shift-up. These observations suggest that a global remodeling of cellular lipids that involves extensive fatty acid exchange between neutral and polar lipid pools represents a novel and important mechanism of adaptation of the parasites to the temperature changes they encounter in their life cycle.

Trypanosoma cruzi: involvement of intracellular calcium in multiplication and differentiation.

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Spermine action on mitochondrial H(+)-ATPase activity and proliferation rate of Trypanosoma cruzi.

Activity of H(+)-ATPase of Trypanosoma cruzi (RA strain) submitochondrial particles is increased in parasites which have a low spermine content caused by growing in a polyamine free medium or in a medium containing inhibitors of polyamine synthesis. Under these conditions, the proliferation rate is markedly decreased. Kinetics of the enzyme inhibition by spermine indicates a non-competitive inhibition. Spermine, through its action on the H(+)-ATPase hydrophobic environment, affects the enzyme activity. Together with the ATPase protein inhibitor, this could be a mechanism regulating ATP levels needed for the parasite proliferation.

Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms.

A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.

Polyamines in Trypanosoma cruzi.

Growth and polyamine content of epimastigotes of Trypanosoma cruzi were studied in the presence of precursors or inhibitors of putrescine synthesis. Arginine and agmatine turned out to be better precursors than ornithine. alpha-D-difluoromethylarginine, an inhibitor of arginine decarboxylase, inhibited cellular proliferation and decreased putrescine, spermidine and spermine contents while that of cadaverine remained unchanged. These effects were reversed both by agmatine and putrescine. alpha-D-difluoromethylornithine, an inhibitor of ornithine decarboxylase did not inhibit growth of parasites grown in a polyamine free medium. These results suggest that epimastigotes need polyamines to grow, and that the parasite are able to synthesize them mainly through the arginine decarboxylase pathway.

An alpha D-globin fragment from Triatoma infestans hindgut stimulates Trypanosoma cruzi adenylyl cyclase and promotes metacyclogenesis.

A peptide from hindguts of the Triatoma infestans, the hematophagous Chagas' insect vector, activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes (proliferative and non-infectious forms) to metacyclic trypomastigotes (non-proliferative and infectious forms). The peptide was purified from hindguts of insects fed two days before with chicken blood. After purification, the peptide showed upon SDS-PAGE a single band of about 10 kDa. The sequence for 20 residues of the amino terminus of this peptide was: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln-Gln-Ala-Trp-Glu-Lys-Ala- Ala-Ser-His. This sequence corresponds to the amino terminus of chicken alpha D-globin. A synthetic peptide with the sequence of the 40 amino acids corresponding to the amino terminus of alpha D-globin, also stimulated T. cruzi adenylyl cyclase activity and promoted metacyclogenesis.

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process.

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129% higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29%) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

Characterization of membrane currents in single smooth muscle cells from the guinea-pig gastric antrum.

1. Smooth muscle cells, enzymatically isolated from the antrum of the guinea-pig stomach, were voltage clamped at room temperature using the whole-cell patch clamp technique. In physiological salt solution (PSS), step depolarization from a holding potential of -90 mV elicited inward calcium current (ICa) followed and superimposed by outward potassium current. 2. Outward current was divided into components depending on the presence of extracellular Ca2+ and others which were not activated as a result of Ca2+ influx. Ca(2+)-dependent components were (1) a fast transient component most likely representing Ca(2+)-activated K+ current (IK(Ca)) immediately triggered by the initial peak of ICa and (2) spontaneous transient outward currents (STOCs) apparently reflecting synchronized opening of IK(Ca) channels by cyclic release of Ca2+ from intracellular stores. Ca2+ influx-independent outward current could be divided into two main components: (1) a transient component (I(to)) showing voltage-dependent activation and inactivation and (2) a non-inactivating component (Ini). 3. I(to) activated with a threshold around -30 mV, was fully available at -90 mV and completely inactivated at -10 mV. The time course of both activation and inactivation of I(to) at different potentials could be described by single exponential functions. Time constants of activation decreased from 35 ms at -10 mV to 10 ms at +40 mV. The time constant of inactivation was about 2 s and only weakly voltage dependent. Time constants for exponentially developing recovery from inactivation of I(to) ranged from 0.1 s at -100 mV to 10 s at -30 mV. I(to) was insensitive to 4-aminopyridine (4-AP, 5 mmol/l), slightly sensitive to tetraethylammonium (TEA, 10 mmol/l), but substantially inhibited by caffeine (10 mmol/l) and Cd2+ (5 mmol/l). Estimates of the single-channel conductance by current fluctuation analysis indicated a small value of about 2.5 pS. 4. The action of TEA on current elicited from a holding potential of -10 mV indicated a major contribution to Ini of a distinct component (Ini,K) that was completely blocked by this substance at a concentration of 10 mmol/l. Ini was almost unaffected by 4-AP (5 mmol/l) and caffeine (10 mmol/l), but strongly suppressed by Cd2+ (5 mmol/l). Current fluctuation analysis of Ini,K gave a value for the single-channel conductance of about 60 pS. 5. Ca2+ inward current was studied in PSS ([Ca2+]o = 2.5 mmol/l) using pipette solution in which K+ was replaced by Cs+ to suppress outward K+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process.

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129

higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29

) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process.

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129

higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29

) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129

higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29

) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

Trypanosoma cruzi: incorporation of macromolecule precursors during the morphogenic process

During the morphogenic process from epimastigote to metacyclic trypomastigote stage changes at molecular levels are produced. In the present work the incorporation of macromolecule precursors L [3H]-leucine, [3H]-thymidine and [3H]-uridine was used to monitor the differences between parasites growing with and without specific stimulation for morphogenesis (SEpi and NEpi, respectively). The peak of maximum [3H]-uridine incorporation was earlier in the SEpi than in NEpi (3 vs 4 days, respectively) reaching 129

higher values. Even at day 2, SEpi already showed values of [3H]-uridine incorporation slightly higher (29

) than the maximum reached by NEpi. One the other hand the peak of L-[3H]-leucine incorporation was 48 h delayed comparing SEpi vs NEpi with quantitatively similar patterns, while no differences were registered for [3H]-thymidine incorporation. Therefore the [3H]-uridine incorporation may constitute an earlier marker of morphogenesis since the process was optically detected starting day at 6.

Suppression of steady membrane currents by acetylcholine in single smooth muscle cells of the guinea-pig gastric fundus.

1. Single smooth muscle cells from the fundus region of the guinea-pig stomach, which showed contractile responses to acetylcholine (ACh) at concentrations greater than or equal to 10(-7) mol/l, were obtained by enzymatic digestion using highly purified collagenase and papain. They were studied by recording membrane currents under voltage clamp with the patch pipette technique in the whole-cell configuration at 25-28 degrees C. 2. By applying voltage jumps from negative holding levels (-70 to -60 mV) to more positive levels, we identified two major activating currents: an initial inward Ca2+ current (ICa) was followed, and partly overlapped, by an outward K+ current (IK). 3. Cholinergic effects on membrane currents were investigated in the range of negative membrane potentials by determining current-voltage relations in the absence of ACh and during its continuous presence in the bathing fluid. 4. ACh induced a decrease in the steady-state conductance which was reversibly blocked by atropine. At physiological external K+ concentration [( K+]o = 6 mmol/l), the reversal potential (Erev) of the current suppressed by ACh (3 x 10(-6) mol/l) was about 20 mV more positive than the calculated K+ equilibrium potential (EK). 5. When [K+]o was increased, Erev was shifted positively; but at each [K+]o, Erev was more positive than EK. 6. Like ACh (10(-6) mol/l), tetraethylammonium (TEA, 1 mmol/l) also suppressed a current with a reversal potential that was, at physiological [K+]o, 20 mV more positive than EK. ACh (10(-5) mol/l) applied in the presence of 1 mmol/l TEA suppressed a pure K+ current (Erev = EK), which was also suppressed by 10 mmol/l TEA. 7. When K+ in the pipette and in the bathing solution was completely replaced by Na+, both ACh (10(-5) mol/l) and TEA (1 mmol/l) caused a reduction of the membrane conductance that appeared to be identical. TEA added to the bathing solution in the presence of ACh did not produce a significant additional conductance decrease. These results did not depend on whether Cl- was present as a charge carrier or not. 8. It is concluded that in fundus muscle of the guinea-pig stomach a major mechanism underlying muscarinic activation is a decrease of a K+ conductance. In addition the results indicate a suppression of a small Na+ conductance which is made up by a population of channels that are also blocked by TEA.

Myogenic basis of motility in the pyloric region of human and canine stomachs.

In vitro gastric motility was investigated in 48 human and 16 canine stomachs by measuring the mechanical activity of isolated muscle strips under auxotonic conditions. For a precise regional differentiation, we recorded the mechanical activity of longitudinal and circular strips from fundus, corpus and antrum, as well as from circular preparations of the inner and outer layer of the pyloric sphincter and from the duodenum. The analysis showed that the anatomical division of the stomach into three distinct regions resulted physiologically in different patterns of contraction in vitro for each region. The fundus exhibited purely tonic spontaneous activity and a tonic contraction pattern after application of acetylcholine whereas the activity in the circular antrum was purely phasic. A combination of tonic and phasic contractions was found in the corpus and longitudinal antrum. A major difference in the basic spontaneous activity pattern was evident between man and dog. A gradient of intrinsic frequency in the stomach from proximal to distal was seen in the dog but not in man. A physiologically distinct area exists in the pyloric region of both species adjacent to the antrum and duodenum. The pyloric ring has its own spontaneous activity (minute-rhythm), reacts to the application of acetylcholine with relatively weak contractions and, unique to the dog, was delineated by histamine-induced maximal contractions. The results provide evidence that the pyloric ring is a distinct organ with specific functional characteristics in its cellular-myogenic structure.