Assessment of the microcirculation is of major importance in understanding the physiology of the vasculature and in assessing te vascular effects of pathological conditions such as diabetes, hypertension and sepsis. Transdermal iontophoresis can be used to non‐invasively introduce vasoactive drugs into the skin. The response to these drugs of the local cutaneous microvasculature can be measured by laser Doppler flowmetry methods. Although these techniques have been used together for over two decades, there are still important methodological issues to be resolved. This work is aimed at optimizing transdermal iontophoresis as a tool for microvascular assessment by focusing on the main methdological issues: non‐specific vasodilatation, drug delivery protocols and analysis of blood flow data. Non‐specific vasodilatation, an increase blood flow during iontophoresis of non‐vasoactive compounds, is an important problem as it interferes with the response to the administered drug. By investigating this effect in healthy volunteers, we found that the extent of the non‐specific response differs between the positive and negative electrode and that it is dependent on the voltage over the skin andon the ionic strength of the vehicle in which the drug is dissolved. We also found that the extent of the non‐specific response could be reduced by applying local anesthetics and by pre‐treatment with antihistamine drugs. These results suggest that non‐specific effects could be mediated by depolarization or hyperpolarisation of cells, triggering neural and histamine related mechanisms that finally lead to vasodilatation of the local microvasculature. To prevent non‐specific effects from occurring during the experiments, our results show that the current strength and the total electric charge during iontophoresis should be limited to 0.02 mA and12 mC, respectively. Furthermore, drug solutions at physiological ionic strengths should be used. Under these conditions, adequate responses to the most commonly used drugs, acetylcholine (ACh) and sodium nitroprusside (SNP), are obtained while no significant non‐specific vasodilatation occurs. The results of our investigations show that blood responses to ACh and SNP applied by a single iontophoretic pulse can well be escribed by conventional dose‐response models, which enables a more powerful analysis and comparison between drugs or possibly patient groups as compared with conventional aalysis methods. Finally, we have incorporated drug transport and physiological response to the local drug concentration during iontophoresis of vasoactve drugs into a single model. Validation of this model using measured responses to ACh and SNP shows that the commonly used assumption that the local drug concentration during iontophoresis is linearly proportional to the electric charge may not be valid. / Mikrocirkulationen, som inbegriper kroppens minsta blodkärl, transporterar syre och näringsämnen till våra celler. Vissa sjukdomar, som diabetes, hjärt‐kärlsjukdom och akut blodförgiftning leder till förändringar hos mikrocirkulationen. Mekanismerna bakom dessa förändringar är delvis okända. Det finns därför ett stort behov av kliniska mättekniker som kan bedöma mikrocirkulationens funktion. Vid jontofores placeras en elektrod tillsammans med ett läkemedel på huden. När en svag elektrisk ström anbringas transporteras läkemedlet ner genom hudlagren. Effekterna av ett kärlaktivt läkemedel som appliceras på detta sätt kan sedan avläsas non‐invasivt med laser Doppler‐teknik. En stor fördel med jontoforesmetoden, förutom att den är non‐invasiv, är att läkemedelsdoserna som tillförs kroppen är mycket små och därmed ger de inte upphov till några systemiska bi‐effekter. I avhandlingen presenteras forskning, vilkas målsättning är att lösa några av de viktiga frågorna kring transdermal jontofores så att tekniken optimeras för att denskall kunna brukas som ett verktyg vid kliniska undersökningar av mikrocirkulationen. Den första delen ägnas ett fenomen som kallas ospecifik vasodilatation. Det uppstår vid jontofores av substanser som är inte kärlaktiv, som vatten och koksaltlösning. Resultaten från dessa försök indikerar att den ospecifika vasodilatationen beror på framför allt spänningen över huden, vilken i sin tur är relaterad till jon‐koncentrationen hos läkemedelslösningen. Vidare registreras att mekanismen bakom den ospecifika vasodilatationen delvis är neuralt medierad genom att de till stor del år att förhindra med hjälp av lokal bedövning. Dessutom leder förbehandling med anti‐histamina läkemedel till minskade ospecifika reaktioner, vilket också indikerar att lokala inflammatoriska processer är inblandande. Den andra delen av avhandlingen ägnas att optimera försöksprotokollen för jontofores. Till att börja med utvecklas ett protokoll som ger ett adekvat läkemedelssvar samtidigt som ospecifika effekter minimeras. Det visar sig är möjligt genom att begränsa strömstyrkan och den elektriska laddningen under jontoforesen och genom att använd läkemedelslösningar som har en fysiologisk jonstyrka. Resultaten visar också att blodflödesförändringen som registreras under jontofores av acetylkolin och natriumnitroprussid kan eskrivas med hjälp av konventionella dos‐responsmodeller, vilket möjliggör en mer exakt analys av det mikrocirkulatoriska svaret samt underlättar jämförelse mellan olika läkemedel elle patientgrupper. Slutligen presenteras en mekanistisk model för det mikrocirkulatoriska svaret vid jontofores. Modellen beskriver läkemedlets transport från elektroden ner genom huden, clearance i huden vilken beror på diffusion och det lokala blodflödet, samt förändringen i blodflöde som sker på grund av läkemedlet. Modellen valideras genom försök på försökspersoner och resultaten visar att förändringarna i blodflödet åstadkommet av acetylklin och natriumnitroprussid med denna modell kan beskrivas på ett exakt sätt. Vidare visar resultaten att det sker en betydande clearance av läkemedel i huden under jontofores. Detta har väsentlig betydelse när man ska uppskatta den lokala jontoforesdosen. / The author changed surname from Droog to Tesselaar in January 2006.

<p>The alpha-2 adrenergic receptor (α₂-AR) subfamily includes three different subtypes, α_2A-, α_2B- and α_2C-AR, all believed to exert their function through heterotrimeric G_i/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α₂-ARs.</p><p>Evidence is provided for a strong G_s-protein coupling capability of the α_2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α_2A- and α_2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α_2A-AR with the corresponding domain of α_2B-AR enrolled the chimeric α_2A/α_2B receptor with functional α_2B-AR properties. Dual G_i and G_s coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α_2B-AR activation is largely dependent on the ratio of G_i- to G_s-protein amounts in the cell. Also G_i- and G_o-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with G_i or G_o and the α_2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through G_o, whereas activation of G_i potentiated the activity. </p><p>α₂-ARs mobilize Ca²⁺ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca²⁺ response of α₂-ARs.</p><p>In conclusion, α₂-ARs can couple to multiple G-proteins, including G_i, G_o and G_s. The cellular response to α₂-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).</p>

The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-, α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α2-ARs. Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α2A-AR with the corresponding domain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties. Dual Gi and Gs coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α2B-AR activation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi- and Go-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gi potentiated the activity. α2-ARs mobilize Ca2+ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca2+ response of α2-ARs. In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. The cellular response to α2-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).

<p>The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. <i>Helicobacter pylori</i> might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of <i>H. pylori</i> on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat.</p><p>A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (<20μm) and discontinuous in the small intestine.</p><p>Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from <i>H. pylori</i> (HPE). In animals with a chronic <i>H. pylori</i> infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. </p><p>HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.</p>

The gastrointestinal mucus gel layer and blood flow are two important mechanisms for protection at the pre-epithelial and sub-epithelial levels, respectively. Helicobacter pylori might circumvent these mechanisms and elicit a chronic inflammatory response with consequent ulcers in the stomach and duodenum. In this thesis, the physical state and properties of the adherent mucus gel layer was studied from the stomach to colon. Furthermore, the acute and chronic effects of H. pylori on the integrity of the mucus gel layer and mucosal blood flow were studied in the anesthetized rat. A translucent mucus gel covers all studied segments of the gastrointestinal tract during fasting conditions, with the thickest layers in the colon and ileum. Carefully applied suction revealed that the mucus gel was a multi-layered structure comprising a firmly adherent layer covering the mucosa, impossible to remove, and a loosely adherent upper layer. The firmly adherent layer was thick and continuous in the corpus (80μm), antrum (154μm) and colon (116μm), but thin (&lt;20μm) and discontinuous in the small intestine. Following mucus removal, a rapid renewal of the loosely adherent layer ensued. The highest rate was observed in the colon with intermediate values in the small intestine. Mucus renewal in the stomach was attenuated on acute luminal application of water extracts from H. pylori (HPE). In animals with a chronic H. pylori infection the mucus renewal rate was unaffected, but the total gastric mucus gel thickness was reduced and the mucus secretory response to luminal acid (pH1) attenuated in the antrum. HPE from type I strains acutely reduced corporal mucosal blood flow, measured with laser-Doppler flowmetry, by approximately 15%. The reduction in blood flow was mediated by a heat stable factor other than VacA and CagA. Inhibition of endogenous nitric oxide production with Nω-nitro-l-arginine augmented the decrease. However, ketotifen, a mast cell stabilizer, completely attenuated the effect of the extract as did the platelet activating factor (PAF) receptor-antagonist, WEB2086, thus depicting a detrimental role for the microvascular actions of PAF.